Maize multidrug resistance-associated protein polynucleotides and methods of use

ABSTRACT

Compositions and methods are provided for modulating the level of phytate in plants. More specifically, the invention relates to methods of modulating the level of phytate utilizing nucleic acids comprising multidrug resistance-associated protein (MRP) nucleotide sequences to modulate the expression of MRP(s) in a plant of interest. The compositions and methods of the invention find use in agriculture for improving the nutritional quality of food and feed by reducing the levels of phytate and/or increasing the levels of non-phytate phosphorus in food and feed. The invention also finds use in reducing the environmental impact of animal waste.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/371,209, filed Feb. 13, 2009, which is a continuation of U.S.application Ser. No. 11/133,075, filed May 19, 2005, which claims thebenefit of U.S. Provisional Application No. 60/572,704, filed May 20,2004, the contents of which are hereby incorporated by reference intheir entirety.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named411724SEQLIST.TXT, created on Nov. 11, 2011, and having a size of 199kilobytes and is filed concurrently with the specification. The sequencelisting contained in this ASCII formatted document is part of thespecification and is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the field of animal nutrition.Specifically, the present invention relates to the identification anduse of genes encoding enzymes involved in the metabolism of phytate inplants and the use of these genes and mutants thereof to reduce thelevels of phytate, and/or increase the levels of non-phytate phosphorusin food or feed.

BACKGROUND OF THE INVENTION

The role of phosphorous in animal nutrition is well recognized.Phosphorus is a critical component of the skeleton, nucleic acids, cellmembranes and some vitamins. Though phosphorous is essential for thehealth of animals, not all phosphorous in feed is bioavailable.

Phytates are the major form of phosphorous in seeds. For example,phytate represents about 60-80% of total phosphorous in corn andsoybean. When seed-based diets are fed to non-ruminants, the consumedphytic acid forms salts with several important mineral nutrients, suchas potassium, calcium, and iron, and also binds proteins in theintestinal tract. These phytate complexes cannot be metabolized bymonogastric animals and are excreted, effectively acting asanti-nutritional factors by reducing the bioavailability of dietaryphosphorous and minerals. Phytate-bound phosphorous in animal excretaalso has a negative environmental impact, contributing to surface andground water pollution.

There have been two major approaches to reducing the negativenutritional and environmental impacts of phytate in seed. The firstinvolves post-harvest interventions, which increase the cost andprocessing time of feed. Post-harvest processing technologies removephytic acid by fermentation or by the addition of compounds, such asphytases.

The second is a genetic approach. One genetic approach involvesdeveloping crop germplasm with heritable reductions in seed phytic acid.While some variability for phytic acid was observed, there was no changein non-phytate phosphorous. Further, only 2% of the observed variationin phytic acid was heritable, whereas 98% of the variation wasattributed to environmental factors. Another genetic approach involvesselecting low phytate lines from a mutagenized population to producegermplasm. Most mutant lines exhibit a loss of function and arepresumably blocked in the phytic acid biosynthetic pathway; therefore,low phytic acid accumulation will likely be a recessive trait. Incertain cases, this approach has revealed that homozygosity forsubstantially reduced phytate can be lethal. Another genetic approach istransgenic technology, which has been used to increase phytase levels inplants. These transgenic plant tissues or seed have been used as dietarysupplements.

The biosynthetic route leading to phytate is complex and not completelyunderstood, and it has been proposed that the production of phytic acidoccurs by one of two possible pathways. One possible pathway involvesthe sequential phosphorylation of Ins(3)P or myo-inositol, leading tothe production of phytic acid. Another possible pathway involveshydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C,followed by the phosphorylation of Ins(1,4,5)P₃ by inositol phosphatekinases. In developing plant seeds, accumulating evidence favors thesequential phosphorylation pathway. Such evidence includes studies ofthe Lpa2 gene, a gene encoding a maize inositol phosphate kinase whichhas multiple kinase activities. The Lpa2 gene has been cloned, and thelpa2 mutation has been shown to impair phytic acid synthesis. Mutantlpa2 seeds accumulate myo-inositol and inositol phosphate intermediates.

The maize low phytic acid 1 mutant (lpa1) was isolated from anEMS-mutagenized population in the early 1990s by USDA scientists.However, the original lpal-1 allele was previously known to have aphenotype of up to 15% loss of seed dry weight, which could translateinto a yield drag if the lpal-1 mutant was used in product development.Since the discovery of lpa1, the gene responsible for the lpa1 mutationhas been sought for two reasons: 1) the mutant has a phenotype of lowphytic acid and high available phosphorus in grain which makes it usefulin animal feeding and phosphorus waste management; and 2) the lpa1mutant does not accumulate myo-inositol phosphate intermediates,indicating that mutation in this locus impairs a critical step in thephytic acid biosynthesis pathway which was previously uncharacterized.

Based on the foregoing, there exists the need to improve the nutritionalcontent of plants, particularly corn and soybean, by increasingnon-phytate phosphorous and reducing seed phytate. Accordingly, it isdesirable to isolate and characterize the Lpa1 gene in order to placethe expression of this gene under tight control so as to produce plantswhich have reduced seed phytate and increased non-phytate phosphorus.

SUMMARY OF THE INVENTION

Compositions and methods are provided for modulating the level ofphytate in plants. More specifically, the invention relates to methodsof modulating the level of phytate utilizing Lpa1 (ZmMRP3) nucleic acidsto produce transformed plants that exhibit decreased expression of atleast one multidrug resistance-associated protein (MRP). Thecompositions and methods of the invention find use in agriculture forimproving the nutritional quality of food and feed by reducing thelevels of phytate and/or increasing the levels of non-phytate phosphorusin food and feed. Thus, the invention finds use in producing food andfeed products as well as in reducing the environmental impact of animalwaste. Also provided are compositions and methods for producing MRPproteins.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B: Alignment of ZmMRP3 (SEQ ID NO: 3) with Pfam consensussequences for ABC transporter (“ABC_tran”; SEQ ID NO: 62) and ABCtransporter transmembrane (“ABC_membrane”; SEQ ID NO: 63) region.

FIG. 2: Diagram of ZmMRP3 and rice OsMRP13 gene structure.

FIG. 3: Phylogenetic comparison of maize, rice and Arabidopsis MRPgenes, showing that maize ZmMRP3, rice OsMRP13 and Arabidopsis AtMRP5are closely related.

FIG. 4A, 4B, 4C, 4D, 4E: cDNA sequence alignment of the maize Lpa1 gene(SEQ ID NO: 2) and its rice homolog OsMRP13 (SEQ ID NO: 6).

FIG. 5A, 5B, 5C: Protein Sequence alignment of maize Lpa1 (ZmMRP3; SEQID NO: 3) with rice and Arabidopsis homologs OsMRP13 (SEQ ID NO: 7) andAtMRP5 (SEQ ID NO: 9). Matches to the consensus are indicated by boldtype; conservative changes are indicated by underlined text.

FIG. 6: Diagram of sample constructs. These sample constructs illustratevarious configurations that can be used in expression cassettes for usein inhibition of expression, for example, for use in hairpin RNAinterference. Sample construct 1 shows a single promoter and fully orpartially complementary sequences of “region 1” and “region 2.” Sampleconstruct 2 illustrates a configuration of two sets of fully orpartially complementary sequences. In this sample construct, “region 1”is fully or partially complementary to “region 2” and “region 3” isfully or partially complementary to “region 4.” Sample construct 3illustrates yet another configuration of two sets of fully or partiallycomplementary sequences; here, too, “region 1” is fully or partiallycomplementary to “region 2” and “region 3” is fully or partiallycomplementary to “region 4.”

DETAILED DESCRIPTION OF THE INVENTION

The invention is drawn to compositions and methods for modulating thelevel of phytate in plants. Compositions of the invention comprisemultidrug resistance-associated proteins (“MRPs”) of the invention(i.e., proteins that have multidrug resistance-associated proteinactivity (“MRP activity”)), polynucleotides that encode them, andassociated noncoding regions as well as fragments and variants of theexemplary disclosed sequences. For example, the disclosed Lpa1polypeptides having amino acid sequences set forth in SEQ ID NOs: 3, 5,7, 9, 11, 13, and 15 are MRPs and therefore have multidrugresistance-associated protein (“MRP”) activity. In particular, thepresent invention provides for isolated polynucleotides comprisingnucleotide sequences set forth in SEQ ID NOs: 1, 2, 4, 6, 8, 10, 12, and14, or encoding the amino acid sequences shown in SEQ ID NOs: 3, 5, 7,9, 11, 13, and 15, and fragments and variants thereof. In addition, theinvention provides polynucleotides comprising the complements of thesenucleotide sequences. Also provided are polypeptides comprising theamino acid sequences shown in SEQ ID NOs: 3, 5, 7, 9, 11, 13, and 15,polypeptides comprising the conserved domains set forth in SEQ ID NOs:16, 17, 18, 19, 20, 21, 22, 23, and 24, fragments and variants thereof,and nucleotide sequences encoding these polypeptides. Compositions ofthe invention also include polynucleotides comprising at least a portionof the promoter sequence set forth in nucleotides 1 to 3134 of SEQ IDNO: 1 as well as polynucleotides comprising other noncoding regions

Thus, the compositions of the invention comprise isolated nucleic acidsthat encode MRP proteins (e.g., Lpa1), fragments and variants thereof,cassettes comprising polynucleotides of the invention, and isolated MRPproteins. The compositions also include nucleic acids comprisingnucleotide sequences which are the complement, or antisense, of theseMRP nucleotide sequences. The invention further provides plants andmicroorganisms transformed with these novel nucleic acids as well asmethods involving the use of such nucleic acids, proteins, andtransformed plants in producing food (including food products) and feedwith reduced phytate and/or increased non-phytate phosphorus levels. Insome embodiments, the transformed plants of the invention and food andfeed produced therefrom have improved nutritional quality due toincreased availability (bioavailability) of nutrients including, forexample, zinc and iron.

In some embodiments, MRP activity is reduced or eliminated bytransforming a maize plant cell with an expression cassette thatexpresses a polynucleotide that inhibits the expression of an MRP enzymesuch as, for example, an Lpa1 polypeptide. The polynucleotide mayinhibit the expression of one or more MRPs directly, by preventingtranslation of the MRP messenger RNA, or indirectly, by encoding apolypeptide that inhibits the transcription or translation of a maizegene encoding an MRP. Methods for inhibiting or eliminating theexpression of a gene in a plant are well known in the art, and any suchmethod may be used in the present invention to inhibit the expression ofone or more maize MRPs. Because MRP activity is difficult to measuredirectly, a decrease in MRP activity can be measured by a decreasedlevel of phytate in a plant or plant part. See, e.g., the workingexamples in the Experimental section.

In accordance with the present invention, the expression of an MRPprotein is inhibited if the transcript or protein level of the MRP isstatistically lower than the transcript or protein level of the same MRPin a plant that has not been genetically modified or mutagenized toinhibit the expression of that MRP. In particular embodiments of theinvention, the transcript or protein level of the MRP in a modifiedplant according to the invention is less than 95%, less than 90%, lessthan 85%, less than 80%, less than 75%, less than 70%, less than 60%,less than 50%, less than 40%, less than 30%, less than 20%, less than10%, or less than 5% of the protein level of the same MRP in a plantthat is not a mutant or that has not been genetically modified toinhibit the expression of that MRP. The expression level of the MRP maybe measured directly, for example, by assaying for the level of MRPexpressed in the cell or plant, or indirectly, for example, by measuringthe amount of phytate in the cell or plant. The activity of an MRPprotein is “eliminated” according to the invention when it is notdetectable by at least one assay method.

In other embodiments of the invention, the activity of one or more MRPsis reduced or eliminated by transforming a plant cell with an expressioncassette comprising a polynucleotide encoding a polypeptide thatinhibits the activity of one or more MRPs. The activity of an MRP isinhibited according to the present invention if the activity of that MRPin the transformed plant or cell is statistically lower than theactivity of that MRP in a plant that has not been genetically modifiedto inhibit the activity of at least one MRP. In particular embodimentsof the invention, an MRP activity of a modified plant according to theinvention is less than 95%, less than 90%, less than 85%, less than 80%,less than 75%, less than 70%, less than 60%, less than 50%, less than40%, less than 30%, less than 20%, less than 10%, or less than 5% ofthat MRP activity in an appropriate control plant that has not beengenetically modified to inhibit the expression of that MRP. Changes inMRP activity may be inferred, for example, by alterations in phytatecontent of a transformed plant or plant cell.

In other embodiments, the activity of an MRP may be reduced oreliminated by disrupting the gene encoding the MRP. The inventionencompasses mutagenized plants that carry at least one mutation in anMRP gene, wherein the at least one mutation reduces expression of an MRPgene or inhibits the activity of an MRP.

Thus, many methods may be used to reduce or eliminate the activity of anMRP. More than one method may be used to reduce the activity of a singleplant MRP. In addition, combinations of methods may be employed toreduce or eliminate the activity of two or more different MRPs.Non-limiting examples of methods of reducing or eliminating theexpression of a plant MRP are given below.

In some embodiments of the present invention, a plant cell istransformed with an expression cassette that is capable of producing apolynucleotide that inhibits the expression of an MRP. The term“expression” as used herein refers to the biosynthesis of a geneproduct, including the transcription and/or translation of said geneproduct. For example, for the purposes of the present invention, anexpression cassette capable of expressing a polynucleotide that inhibitsthe expression of at least one maize MRP is an expression cassettecapable of producing an RNA molecule that inhibits the transcriptionand/or translation of at least one maize MRP.

“Expression” generally refers to the transcription and/or translation ofa coding region of a DNA molecule, messenger RNA, or other nucleic acidmolecule to produce the encoded protein or polypeptide. In othercontexts, “expression” refers to the transcription of RNA from anexpression cassette, such as, for example, the transcription of ahairpin construct from an expression cassette for use in hpRNAinterference.

“Coding region” refers to the portion of a messenger RNA (or thecorresponding portion of another nucleic acid molecule such as a DNAmolecule) which encodes a protein or polypeptide. “Noncoding region”refers to all portions of a messenger RNA or other nucleic acid moleculethat are not a coding region, including, for example, the promoterregion, 5′ untranslated region (“UTR”), and/or 3′ UTR.

Some examples of polynucleotides and methods that inhibit the expressionof an MRP are given below. While specific examples are given below, avariety of methods are known in the art by which it is possible toinhibit expression. While the invention is not bound by any particulartheory of operation or mechanism of action, the invention provides theexemplary nucleotide and protein sequences disclosed herein and therebyprovides a variety of methods by which expression can be inhibited. Forexample, fragments of noncoding region can be used to make constructsthat inhibit expression of an MRP; such fragments can include portionsof the promoter region or portions of the 3′ noncoding region (i.e., the3′ UTR).

In some embodiments of the invention, inhibition of the expression of anMRP may be obtained by sense suppression or cosuppression. Forcosuppression, an expression cassette is designed to express an RNAmolecule corresponding to all or part of a messenger RNA encoding an MRPin the “sense” orientation. Overexpression of the RNA molecule canresult in reduced expression of the native gene. Accordingly, multipleplant lines transformed with the cosuppression expression cassette arescreened to identify those that show suitable inhibition of MRPexpression.

The polynucleotide used for cosuppression or other methods to inhibitexpression may correspond to all or part of the sequence encoding theMRP, all or part of the 5′ and/or 3′ untranslated region of an MRPtranscript, or all or part of both the coding region and theuntranslated regions of a transcript encoding MRP. A polynucleotide usedfor cosuppression or other gene silencing methods may share 99%, 98%,97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 85%, 80%, or lesssequence identity with the target sequence. When portions of thepolynucleotides are used to disrupt the expression of the target gene,generally, sequences of at least 15, 20, 30, 35, 40, 45, 50, 60, 70, 80,90, 100, 200, 300, 400, 450, 500, 550, 600, 650, 700, 750, 800, or 900nucleotides or 1 kb or greater may be used. In some embodiments wherethe polynucleotide comprises all or part of the coding region for theMRP, the expression cassette is designed to eliminate the start codon ofthe polynucleotide so that no protein product will be transcribed. Inthis manner, an expression cassette may cause permanent modification ofthe coding and/or noncoding region of an endogenous gene.

Thus, in some embodiments, for example, the polynucleotide used forcosuppression or another method to inhibit expression will comprise asequence selected from a particular region of the coding and/ornoncoding region. That is, the polynucleotide will comprise a sequenceor the complement of a sequence selected from the region betweennucleotides 1 and 5139 of the sequence set forth in SEQ ID NO: 2, orselected from the region with a first endpoint at nucleotide 1, 150,250, 400, 550, 700, 850, 1000, 1150, 1300, 1450, 1600, 1750, 1900, 2050,2200, 2350, 2500, 2650, 2800, 2950, 3100, 3250, 3400, 3550, 3700, 3850,4000, 4150, 4300, 4450, 4600, 4750, 4900, 5050, or 5139 and a secondendpoint at nucleotide 244, 400, 550, 700, 850, 1000, 1150, 1300, 1450,1600, 1750, 1900, 2050, 2200, 2350, 2500, 2650, 2800, 2950, 3100, 3250,3400, 3550, 3700, 3850, 4000, 4150, 4300, 4450, 4600, 4750, 4900, 5050,or 5139. As discussed elsewhere herein, fragments and/or variants of theexemplary disclosed sequences may also be used.

In some embodiments, for example, the polynucleotide will comprise asequence or the complement of a sequence selected from the regionbetween nucleotides 1 and 3134 of the sequence set forth in SEQ ID NO:1,or selected from the region with a first endpoint at nucleotide 1, 150,400, 550, 700, 850, 1000, 1150, 1300, 1450, 1600, 1750, 1900, 2050,2200, 2350, 2500, 2650, 2800, 2950, or 3134, and a second endpoint atnucleotide 1, 150, 400, 550, 700, 850, 1000, 1150, 1300, 1450, 1600,1750, 1900, 2050, 2200, 2350, 2500, 2650, 2800, 2950, or 3134. Where anoncoding region is used for cosuppression or other method to inhibitexpression, it may be advantageous to use a noncoding region thatcomprises CpG islands (see, e.g., Tariq et al. (2004) Trends Genet. 20:244-251). As discussed elsewhere herein, variants and/or fragments ofthe exemplary disclosed sequences may also be used.

In some embodiments, for example, the polynucleotide will comprise asequence or the complement of a sequence selected from the regionbetween nucleotides 1 and 5123 of the sequence set forth in SEQ ID NO:6,or selected from the region with a first endpoint at nucleotide 1, 150,300, 450, 550, 700, 850, 1000, 1150, 1300, 1450, 1600, 1750, 1900, 2050,2200, 2350, 2500, 2650, 2800, 2950, 3100, 3250, 3400, 3550, 3700, 3850,4000, 4150, 4300, 4450, 4600, 4750, 4900, or 5123, and a second endpointat nucleotide 1, 150, 300, 450, 550, 700, 850, 1000, 1150, 1300, 1450,1600, 1750, 1900, 2050, 2200, 2350, 2500, 2650, 2800, 2950, 3100, 3250,3400, 3550, 3700, 3850, 4000, 4150, 4300, 4450, 4600, 4750, 4900, or5123. As discussed elsewhere herein, variants and/or fragments of theexemplary disclosed sequences may also be used.

In some embodiments, for example, the polynucleotide will comprise asequence or the complement of a sequence selected from the regionbetween nucleotides 1 and 1350 of the sequence set forth in SEQ IDNO:10, or selected from the region with a first endpoint at nucleotide1, 150, 300, 450, 550, 700, 850, 1000, 1150, 1300, or 1350, and a secondendpoint at nucleotide 1, 150, 300, 450, 550, 700, 850, 1000, 1150,1300, or 1350. As discussed elsewhere herein, variants and/or fragmentsof the exemplary disclosed sequences may also be used.

In some embodiments, for example, the polynucleotide will comprise asequence or the complement of a sequence selected from the regionbetween nucleotides 1 and 465 of the sequence set forth in SEQ ID NO:12,or selected from the region with a first endpoint at nucleotide 1, 150,300, 450, or 465, and a second endpoint at nucleotide 1, 150, 300, 450,or 465. As discussed elsewhere herein, variants and/or fragments of theexemplary disclosed sequences may also be used.

In some embodiments, for example, the polynucleotide will comprise asequence or the complement of a sequence selected from the regionbetween nucleotides 1 and 556 of the sequence set forth in SEQ ID NO:71,or selected from the region with a first endpoint at nucleotide 1, 150,300, 450, or 556, and a second endpoint at nucleotide 1, 150, 300, 450,or 556. As discussed elsewhere herein, variants and/or fragments of theexemplary disclosed sequences may also be used.

Cosuppression may be used to inhibit the expression of plant genes toproduce plants having undetectable protein levels for the proteinsencoded by these genes. See, for example, Broin et al. (2002) Plant Cell14: 1417-1432. Cosuppression may also be used to inhibit the expressionof multiple proteins in the same plant. See, for example, U.S. Pat. No.5,942,657. Methods for using cosuppression to inhibit the expression ofendogenous genes in plants are described in Flavell et al. (1994) Proc.Natl. Acad. Sci. USA 91: 3490-3496; Jorgensen et al. (1996) Plant Mol.Biol. 31: 957-973; Johansen and Carrington (2001) Plant Physiol. 126:930-938; Broin et al. (2002) Plant Cell 14: 1417-1432; Stoutjesdijk etat (2002) Plant Physiol. 129: 1723-1731; Yu et al. (2003) Phytochemistry63: 753-763; and U.S. Pat. Nos. 5,034,323; 5,283,184; and 5,942,657;each of which is herein incorporated by reference. The efficiency ofcosuppression may be increased by including a poly-dT region in theexpression cassette at a position 3′ to the sense sequence and 5′ of thepolyadenylation signal. See, e.g., U.S. Patent Publication No.20020048814, herein incorporated by reference. Typically, such anucleotide sequence has substantial sequence identity to the sequence ofthe transcript of the endogenous gene, for example, greater than about65%, 80%, 85%, 90%, 95%, or more sequence identity. See, U.S. Pat. Nos.5,283,184 and 5,034,323, herein incorporated by reference.

In some embodiments of the invention, inhibition of the expression ofthe MRP may be obtained by antisense suppression. For antisensesuppression, the expression cassette is designed to express an RNAmolecule complementary to all or part of a messenger RNA comprising aregion encoding the MRP. Overexpression of the antisense RNA moleculecan result in reduced expression of the native gene. Accordingly,multiple plant lines transformed with the antisense suppressionexpression cassette are screened to identify those that show thegreatest inhibition of MRP expression.

The polynucleotide for use in antisense suppression may correspond toall or part of the complement of the sequence encoding the MRP, all orpart of the complement of the 5′ and/or 3′ untranslated region of theMRP transcript, or all or part of the complement of both the codingsequence and the untranslated regions of a transcript encoding the MRP.In addition, the antisense polynucleotide may be fully complementary(i.e., 100% identical to the complement of the target sequence) orpartially complementary (i.e., less than 100% identical to thecomplement of the target sequence) to the target sequence. That is, anantisense polynucleotide may share 99%, 98%, 97%, 96%, 95%, 94%, 93%,92%, 91%, 90%, 89%, 88%, 87%, 85%, 80%, or less sequence identity withthe target sequence. Antisense suppression may be used to inhibit theexpression of multiple proteins in the same plant. See, for example,U.S. Pat. No. 5,942,657. Furthermore, portions of the antisensenucleotides may be used to disrupt the expression of the target gene.Generally, sequences of at least 30, 40, 50, 60, 70, 80, 90, 100, 200,300, 400, 450, 500, or 550 nucleotides or greater may be used.

Methods for using antisense suppression to inhibit the expression ofendogenous genes in plants are described, for example, in Liu et at(2002) Plant Physiol. 129:1732-1743 and U.S. Pat. Nos. 5,759,829 and5,942,657, each of which is herein incorporated by reference. Efficiencyof antisense suppression may be increased by including a poly-dT regionin the expression cassette at a position 3′ to the antisense sequenceand 5′ of the polyadenylation signal. See, U.S. Patent Publication No.20020048814, herein incorporated by reference.

In some embodiments of the invention, inhibition of the expression of anMRP may be obtained by double-stranded RNA (dsRNA) interference. FordsRNA interference, a sense RNA molecule like that described above forcosuppression and an antisense RNA molecule that is fully or partiallycomplementary to the sense RNA molecule are expressed in the same cell,resulting in inhibition of the expression of the correspondingendogenous messenger RNA.

Expression of the sense and antisense molecules can be accomplished bydesigning the expression cassette to comprise both a sense sequence andan antisense sequence. Alternatively, separate expression cassettes maybe used for the sense and antisense sequences. Multiple plant linestransformed with the dsRNA interference expression cassette orexpression cassettes are then screened to identify plant lines that showthe greatest inhibition of MRP expression. Methods for using dsRNAinterference to inhibit the expression of endogenous plant genes aredescribed in Waterhouse et al. (1998) Proc. Natl. Acad. Sci. USA 95:13959-13964, Liu et al. (2002) Plant Physiol. 129: 1732-1743, and WO99/49029, WO 99/53050, WO 99/61631, and WO 00/49035; each of which isherein incorporated by reference.

In some embodiments of the invention, inhibition of the expression ofone or more MRPs may be obtained by hairpin RNA (hpRNA) interference orintron-containing hairpin RNA (ihpRNA) interference. These methods arehighly efficient at inhibiting the expression of endogenous genes. See,Waterhouse and Helliwell (2003) Nat. Rev. Genet. 4: 29-38 and thereferences cited therein. These methods can make use of either codingregion sequences or promoter or regulatory region sequences.

For hpRNA interference, the expression cassette is designed to expressan RNA molecule that hybridizes with itself to form a hairpin structurethat comprises a single-stranded loop or “spacer” region and abase-paired stem. In some embodiments, the base-paired stem regioncomprises a sense sequence corresponding to all or part of theendogenous messenger RNA encoding the gene whose expression is to beinhibited, and an antisense sequence that is fully or partiallycomplementary to the sense sequence. The antisense sequence may belocated “upstream” of the sense sequence (i.e., the antisense sequencemay be closer to the promoter driving expression of the hairpin RNA thanthe sense sequence). In some embodiments, the base-paired stem regioncomprises a first portion of a noncoding region such as a promoter and asecond portion of the noncoding region that is in inverted orientationrelative to the first portion and that is fully or partiallycomplementary to the first portion. In some embodiments, the base-pairedstem region comprises a first portion and a second portion which arefully or partially complementary to each other but which comprise bothcoding and noncoding regions.

In some embodiments, the expression cassette comprises more than onebase-paired “stem” region; that is, the expression cassette comprisessequences from different coding and/or noncoding regions which have thepotential to form more than one base-paired “stem” region, for example,as diagrammed in FIG. 6 (construct 2 and construct 3). Where more thanone base-paired “stem” region is present in an expression cassette, the“stem” regions may flank one another as diagrammed in FIG. 6 (construct3) or may be in some other configuration (for example, as diagrammed inFIG. 6 (construct 2)). That is, for example, an expression cassette maycomprise more than one combination of promoter and complementarysequences as shown in FIG. 6 (construct 1), and each such combinationmay be driven by a separate promoter. One of skill will be able tocreate and test a variety of configurations to determine the optimalconstruct for use in this or any other method for inhibition ofexpression.

Thus, the base-paired stem region of the molecule generally determinesthe specificity of the RNA interference. The sense sequence and theantisense sequence (or first and second portion of the noncoding region)are generally of similar lengths but may differ in length. Thus, thesesequences may be portions or fragments of at least 10, 19, 20, 30, 50,70, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340,360, 380, 400, 500, 600, 700, 800, 900 nucleotides in length, or atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 kb in length. The loop region ofthe expression cassette may vary in length. Thus, the loop region may beat least 100, 200, 300, 400, 500, 600, 700, 800, 900 nucleotides inlength, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 kb in length. Insome embodiments, the loop region comprises an intron such as, forexample, the Adh1 intron.

hpRNA molecules are highly efficient at inhibiting the expression ofendogenous genes, and the RNA interference they induce is inherited bysubsequent generations of plants. See, for example, Chuang andMeyerowitz (2000) Proc. Natl. Acad. Sci. USA 97: 4985-4990; Stoutjesdijket al. (2002) Plant Physiol. 129: 1723-1731; and Waterhouse andHelliwell (2003) Nat. Rev. Genet. 4: 29-38. Methods for using hpRNAinterference to inhibit or silence the expression of genes aredescribed, for example, in Chuang and Meyerowitz (2000) Proc. Natl.Acad. Sci. USA 97: 4985-4990; Stoutjesdijk et al. (2002) Plant Physiol.129: 1723-1731; Waterhouse and Helliwell (2003) Nat. Rev. Genet. 4:29-38; Pandolfini et al. BMC Biotechnology 3: 7, and U.S. PatentPublication No. 20030175965; each of which is herein incorporated byreference. A transient assay for the efficiency of hpRNA constructs tosilence gene expression in vivo has been described by Panstruga et al.(2003) Mol. Biol. Rep. 30: 135-140, herein incorporated by reference.

For ihpRNA, the interfering molecules have the same general structure asfor hpRNA (including the same sizes of sense sequences and antisensesequences), but the RNA molecule additionally comprises an intron in theloop or “spacer” region that is capable of being spliced in the cell inwhich the ihpRNA is expressed. The use of an intron minimizes the sizeof the loop in the hairpin RNA molecule following splicing, and thisincreases the efficiency of interference. See, for example, Smith et al.(2000) Nature 407: 319-320 (which demonstrated 100% suppression ofendogenous gene expression using ihpRNA-mediated interference). Methodsfor using ihpRNA interference to inhibit the expression of endogenousplant genes are described, for example, in Smith et al. (2000) Nature407: 319-320; Wesley et al. (2001) Plant J. 27: 581-590; Wang andWaterhouse (2001) Curr. Opin. Plant Biol. 5: 146-150; Waterhouse andHelliwell (2003) Nat. Rev. Genet. 4: 29-38; Helliwell and Waterhouse(2003) Methods 30: 289-295, and U.S. Patent Publication No. 20030180945,each of which is herein incorporated by reference.

The expression cassette for hpRNA interference may also be designed suchthat the sense sequence and the antisense sequence do not correspond toan endogenous RNA. In this embodiment, the sense and antisense sequenceflank a loop sequence that comprises a nucleotide sequence correspondingto all or part of the endogenous messenger RNA of the target gene. Thus,in this embodiment, it is the loop region that determines thespecificity of the RNA interference. See, for example, WO 02/00904,herein incorporated by reference.

Transcriptional gene silencing (TGS) may be accomplished through use ofhpRNA constructs wherein the inverted repeat of the hairpin sharessequence identity with the promoter region of a gene to be silenced.Processing of the hpRNA into short RNAs which can interact with thehomologous promoter region may trigger degradation or methylation toresult in silencing (Aufsatz et al. (2002) Proc. Nat'l. Acad. Sci. USA99 (Suppl. 4): 16499-16506; Mette et al. (2000) EMBO J. 19(19):5194-5201). As the invention is not bound by a particular mechanism ormode of operation, a decrease in expression may also be achieved byother mechanisms.

Amplicon expression cassettes comprise a plant virus-derived sequencethat contains all or part of the target gene but generally not all ofthe genes of the native virus. The viral sequences present in thetranscription product of the expression cassette allow the transcriptionproduct to direct its own replication. The transcripts produced by theamplicon may be either sense or antisense relative to the targetsequence (i.e., the messenger RNA for MRP). Methods of using ampliconsto inhibit the expression of endogenous plant genes are described, forexample, in Angell and Baulcombe (1997) EMBO J. 16: 3675-3684, Angelland Baulcombe (1999) Plant J. 20: 357-362, and U.S. Pat. No. 6,646,805,each of which is herein incorporated by reference.

In some embodiments, the polynucleotide expressed by the expressioncassette of the invention is catalytic RNA or has ribozyme activityspecific for the messenger RNA of MRP. Thus, the polynucleotide causesthe degradation of the endogenous messenger RNA, resulting in reducedexpression of the MRP. This method is described, for example, in U.S.Pat. No. 4,987,071, herein incorporated by reference.

In some embodiments of the invention, inhibition of the expression ofone or more MRPs may be obtained by RNA interference by expression of agene encoding a micro RNA (miRNA). miRNAs are regulatory agentsconsisting of about 22 ribonucleotides. miRNAs are highly efficient atinhibiting the expression of endogenous genes. See, for example Javieret al. (2003) Nature 425: 257-263, herein incorporated by reference.

For miRNA interference, the expression cassette is designed to expressan RNA molecule that is modeled on an endogenous miRNA gene. The miRNAgene encodes an RNA that forms a hairpin structure containing a22-nucleotide sequence that is complementary to another endogenous gene(target sequence). For suppression of MRP expression, the 22-nucleotidesequence is selected from an MRP transcript sequence and contains 22nucleotides of said MRP sequence in sense orientation and 21 nucleotidesof a corresponding antisense sequence that is complementary to the sensesequence. miRNA molecules are highly efficient at inhibiting theexpression of endogenous genes, and the RNA interference they induce isinherited by subsequent generations of plants.

In one embodiment, the polynucleotide encodes a zinc finger protein thatbinds to a gene encoding an MRP resulting in reduced expression of thegene. In particular embodiments, the zinc finger protein binds to aregulatory region of an MRP gene. In other embodiments, the zinc fingerprotein binds to a messenger RNA encoding an MRP and prevents itstranslation. Methods of selecting sites for targeting by zinc fingerproteins have been described, for example, in U.S. Pat. No. 6,453,242,and methods for using zinc finger proteins to inhibit the expression ofgenes in plants are described, for example, in U.S. Patent PublicationNo. 20030037355; each of which is herein incorporated by reference.

In some embodiments of the invention, the polynucleotide encodes anantibody that binds to at least one maize MRP and reduces the phytatelevel of the plant. In another embodiment, the binding of the antibodyresults in increased turnover of the antibody-MRP complex by cellularquality control mechanisms. The expression of antibodies in plant cellsand the inhibition of molecular pathways by expression and binding ofantibodies to proteins in plant cells are well known in the art. See,for example, Conrad and Sonnewald (2003) Nature Biotech. 21: 35-36,incorporated herein by reference. In other embodiments of the invention,the polynucleotide encodes a polypeptide that specifically inhibits theMRP activity of a maize MRP, i.e., an MRP inhibitor.

In some embodiments of the present invention, the activity of an MRP isreduced or eliminated by disrupting the gene encoding the MRP. The geneencoding the MRP may be disrupted by any method known in the art. Forexample, in one embodiment, the gene is disrupted by transposon tagging.In another embodiment, the gene is disrupted by mutagenizing maizeplants using random or targeted mutagenesis, and selecting for plantsthat have reduced MRP activity.

In one embodiment of the invention, transposon tagging is used to reduceor eliminate the activity of one or more MRPs. Transposon taggingcomprises inserting a transposon within an endogenous MRP gene to reduceor eliminate expression of the MRP. “MRP gene” is intended to mean thegene that encodes an MRP protein according to the invention.

In this embodiment, the expression of one or more MRPs is reduced oreliminated by inserting a transposon within a regulatory region orcoding region of the gene encoding the MRP. A transposon that is withinan exon, intron, 5′ or 3′ untranslated sequence, a promoter, or anyother regulatory sequence of an MRP gene may be used to reduce oreliminate the expression and/or activity of the encoded MRP.

Methods for the transposon tagging of specific genes in plants are wellknown in the art. See, for example, Maes et al. (1999) Trends Plant Sci.4: 90-96; Dharmapuri and Sonti (1999) FEMS Microbiol. Lett. 179: 53-59;Meissner et al. (2000) Plant J. 22: 265-274; Phogat et al. (2000) J.Biosci. 25: 57-63; Walbot (2000) Curr. Opin. Plant Biol. 2: 103-107; Gaiet al. (2000) Nucleic Acids Res. 28: 94-96; Fitzmaurice et al. (1999)Genetics 153: 1919-1928. In addition, the TUSC process for selecting Muinsertions in selected genes has been described in Bensen et al. (1995)Plant Cell 7: 75-84; Mena et al. (1996) Science 274: 1537-1540; and U.S.Pat. No. 5,962,764; each of which is herein incorporated by reference.

Additional methods for decreasing or eliminating the expression ofendogenous genes in plants are also known in the art and can besimilarly applied to the instant invention. These methods include otherforms of mutagenesis, such as ethyl methanesulfonate-inducedmutagenesis, deletion mutagenesis, and fast neutron deletion mutagenesisused in a reverse genetics sense (with PCR) to identify plant lines inwhich the endogenous gene has been deleted. For examples of thesemethods see Ohshima et al. (1998) Virology 243: 472-481; Okubara et al.(1994) Genetics 137: 867-874; and Quesada et al. (2000) Genetics 154:421-436; each of which is herein incorporated by reference. In addition,a fast and automatable method for screening for chemically inducedmutations, TILLING (Targeting Induced Local Lesions In Genomes), usingdenaturing HPLC or selective endonuclease digestion of selected PCRproducts is also applicable to the instant invention. See McCallum etal. (2000) Nat. Biotechnol. 18: 455-457, herein incorporated byreference.

Mutations that impact gene expression or that interfere with thefunction of the encoded protein are well known in the art. Insertionalmutations in gene exons usually result in null-mutants. Mutations inconserved residues are particularly effective in inhibiting the MRPactivity of the encoded protein. Conserved residues of plant MRPssuitable for mutagenesis with the goal to eliminate MRP activity aredescribed herein, for example in the conserved domains set forth in SEQID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24. Such mutants can beisolated according to well-known procedures, and mutations in differentMRP loci can be stacked by genetic crossing. See, for example, Gruis etal. (2002) Plant Cell 14: 2863-2882.

In another embodiment of this invention, dominant mutants can be used totrigger RNA silencing due to gene inversion and recombination of aduplicated gene locus. See, for example, Kusaba et al. (2003) Plant Cell15: 1455-1467.

The invention encompasses additional methods for reducing or eliminatingthe activity of one or more MRPs. Examples of other methods for alteringor mutating a genomic nucleotide sequence in a plant are known in theart and include, but are not limited to, the use of chimeric vectors,chimeric mutational vectors, chimeric repair vectors, mixed-duplexoligonucleotides, self-complementary oligonucleotides, andrecombinogenic oligonucleobases. Such vectors and methods of use areknown in the art. See, for example, U.S. Pat. Nos. 5,565,350; 5,731,181;5,756,325; 5,760,012; 5,795,972; and 5,871,984; each of which are hereinincorporated by reference. See also, WO 98/49350, WO 99/07865, WO99/25821, and Beetham et al. (1999) Proc. Natl. Acad. Sci. USA 96:8774-8778; each of which is herein incorporated by reference. Othermethods of suppressing expression of a gene involve promoter-basedsilencing. See, for example, Mette et al. (2000) EMBO J. 19: 5194-5201;Sijen et al. (2001) Curr. Biol. 11: 436-440; Jones et al. (2001) Curr.Biol. 11: 747-757.

Where polynucleotides are used to decrease or inhibit MRP activity, itis recognized that modifications of the exemplary sequences disclosedherein may be made as long as the sequences act to decrease or inhibitexpression of the corresponding mRNA. Thus, for example, polynucleotideshaving at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the exemplarysequences disclosed herein may be used. Furthermore, portions orfragments of the exemplary sequences or portions or fragments ofpolynucleotides sharing a particular percent sequence identity to theexemplary sequences may be used to disrupt the expression of the targetgene. Generally, fragments or sequences of at least 10, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70,80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 250, 260, 280, 300, 350,400, 450, 500, 600, 700, 800, 900, 1000, or more contiguous nucleotides,or greater may be used. It is recognized that in particular embodiments,the complementary sequence of such sequences may be used. For example,hairpin constructs comprise both a sense sequence fragment and acomplementary, or antisense, sequence fragment corresponding to the geneof interest. Antisense constructs may share less than 100% sequenceidentity with the gene of interest, and may comprise portions orfragments of the gene of interest, so long as the object of theembodiment is achieved, i.e., so long as expression of the gene ofinterest is decreased.

Accordingly, the methods of the invention include methods for modulatingthe levels of endogenous transcription and/or gene expression bytransforming plants with antisense or sense constructs to produce plantswith reduced levels of phytate. In some embodiments, such modificationswill alter the amino acid sequence of the proteins encoded by thegenomic sequence as to reduce or eliminate the activity of a particularendogenous gene, such as MRP, in a plant or part thereof, for example,in a seed.

Furthermore, it is recognized that the methods of the invention mayemploy a nucleotide construct that is capable of directing, in atransformed plant, the expression of at least one protein, or thetranscription of at least one RNA, such as, for example, an antisenseRNA that is complementary to at least a portion of an mRNA. Typicallysuch a nucleotide construct is comprised of a coding sequence for aprotein or an RNA operably linked to 5′ and 3′ transcriptionalregulatory regions. Alternatively, it is also recognized that themethods of the invention may employ a nucleotide construct that is notcapable of directing, in a transformed plant, the expression of aprotein or transcription of an RNA.

In addition, it is recognized that methods of the present invention donot depend on the incorporation of the entire nucleotide construct intothe genome, only that the plant or cell thereof is altered as a resultof the introduction of the nucleotide construct into a cell. In oneembodiment of the invention, the genome may be altered following theintroduction of the nucleotide construct into a cell. For example, thenucleotide construct, or any part thereof, may incorporate into thegenome of the plant. Alterations to the genome of the present inventioninclude, but are not limited to, additions, deletions, and substitutionsof nucleotides in the genome. While the methods of the present inventiondo not depend on additions, deletions, or substitutions of anyparticular number of nucleotides, it is recognized that such additions,deletions, or substitutions comprise at least one nucleotide.

The use of the term “nucleotide constructs” herein is not intended tolimit the present invention to nucleotide constructs comprising DNA.Those of ordinary skill in the art will recognize that nucleotideconstructs, particularly polynucleotides and oligonucleotides, comprisedof ribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides may also be employed in the methods disclosedherein. Thus, the nucleotide constructs of the present inventionencompass all nucleotide constructs that can be employed in the methodsof the present invention for transforming plants including, but notlimited to, those comprised of deoxyribonucleotides, ribonucleotides,and combinations thereof. Such deoxyribonucleotides and ribonucleotidesinclude both naturally occurring molecules and synthetic analogues. Thenucleotide constructs of the invention also encompass all forms ofnucleotide constructs including, but not limited to, single-strandedforms, double-stranded forms, hairpins, stem-and-loop structures, andthe like.

The invention encompasses isolated or substantially purified nucleicacid or protein compositions. An “isolated” or “purified” nucleic acidmolecule or protein, or biologically active portion thereof, issubstantially or essentially free from components that normallyaccompany or interact with the nucleic acid molecule or protein as foundin its naturally occurring environment. Thus, an isolated or purifiednucleic acid molecule or protein is substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. Preferably, an “isolated” nucleic acid is freeof sequences (preferably protein encoding sequences) that naturallyflank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends ofthe nucleic acid) in the genomic DNA of the organism from which thenucleic acid is derived. For example, in various embodiments, theisolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturallyflank the nucleic acid molecule in genomic DNA of the cell from whichthe nucleic acid is derived. A protein that is substantially free ofcellular material includes preparations of protein having less thanabout 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein.When the protein of the invention or biologically active portion thereofis recombinantly produced, preferably culture medium represents lessthan about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemicalprecursors or non-protein-of-interest chemicals.

By “modulating” or “modulate” as used herein is intended that the levelor amount of a product is increased or decreased in accordance with thegoal of the particular embodiment. For example, if a particularembodiment were useful for producing purified MRP enzyme, it would bedesirable to increase the amount of MRP protein produced. As anotherexample, if a particular embodiment were useful for decreasing theamount of phytate in a transgenic plant, it would be desirable todecrease the amount of MRP protein expressed by the plant.

The article “a” and “an” are used herein to refer to one or more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “an element” means one or more element.

Throughout the specification the word “comprising,” or variations suchas “comprises,” will be understood to imply the inclusion of a statedelement, integer or step, or group of elements, integers or steps, butnot the exclusion of any other element, integer or step, or group ofelements, integers or steps.

Fragments and/or variants of the disclosed polynucleotides and proteinsencoded thereby are also encompassed by the present invention. By“fragment” is intended a portion of the polynucleotide or a portion ofthe nucleotide sequence and hence protein encoded thereby, if any.Fragments of a nucleotide sequence may encode protein fragments thatretain the biological activity of the native protein and hence have MRPactivity. Alternatively, fragments of a nucleotide sequence that areuseful as hybridization probes or in sense or antisense suppressiongenerally do not encode fragment proteins retaining biological activity.Thus, fragments of a nucleotide sequence may range in length from atleast about 20 nucleotides, about 50 nucleotides, about 100 nucleotides,and up to the full-length nucleotide sequence encoding the proteins ofthe invention.

A fragment of an MRP nucleotide sequence that encodes a biologicallyactive portion of an MRP protein of the invention will encode at least15, 25, 30, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, or 1500contiguous amino acids, or up to the total number of amino acids presentin a full-length MRP protein of the invention (for example, 1510 aminoacids for SEQ ID NO: 3). Fragments of an MRP nucleotide sequence thatare useful in non-coding embodiments, for example, as PCR primers or forsense or antisense suppression, generally need not encode a biologicallyactive portion of an MRP protein. A fragment of an MRP polypeptide ofthe invention will contain at least 15, 25, 30, 50, 100, 150, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950,1000, 1100, 1200, 1300, 1400, or 1500 contiguous amino acids, or up tothe total number of amino acids present in a full-length MRP protein ofthe invention (for example, 1510 amino acids for SEQ ID NO: 3).

Thus, a fragment of an MRP nucleotide sequence may encode a biologicallyactive portion of an MRP protein, or it may be a fragment that can beused, for example, as a hybridization probe or in sense or antisensesuppression using methods disclosed herein and known in the art. Abiologically active portion of an MRP protein can be prepared byisolating a portion of one of the MRP polynucleotides of the invention,expressing the encoded portion of the MRP protein (e.g., by recombinantexpression in vitro), and assessing the activity of the encoded portionof the MRP protein. Nucleic acid molecules that are fragments orportions of an MRP polynucleotide comprise at least 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 50, 75, 100, 150, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1,000, 1,100, 1,200,1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 3,000, 4,000, or5,000 contiguous nucleotides, or up to the number of nucleotides presentin a full-length MRP polynucleotide disclosed herein (for example, 5139nucleotides for SEQ ID NO: 2).

“Variants” is intended to mean substantially similar sequences. Forpolynucleotides, a variant comprises a deletion and/or addition at oneor more nucleotides at one or more internal sites within the nativepolynucleotide and/or a substitution of one or more nucleotides at oneor more sites in the native polynucleotide. As used herein, a “native”polypeptide or polynucleotide comprises a naturally occurring amino acidsequence or nucleotide sequence. For polynucleotides, conservativevariants include those sequences that, because of the degeneracy of thegenetic code, encode the amino acid sequence of one of the MRPpolypeptides of the invention. Naturally occurring allelic variants suchas these can be identified with the use of well-known molecular biologytechniques, as, for example, with polymerase chain reaction (PCR) andhybridization techniques as outlined below. Variant polynucleotides alsoinclude synthetically-derived polynucleotides, such as those generated,for example, by using site-directed mutagenesis but which still encodean MRP protein of the invention. Generally, variants of a particularpolynucleotide of the invention will have at least about 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity to that particularpolynucleotide as determined by sequence alignment programs andparameters described elsewhere herein.

Variants of a particular polynucleotide of the invention (i.e., thereference polynucleotide) can also be evaluated by comparison of thepercent sequence identity between the polypeptide encoded by a variantpolynucleotide and the polypeptide encoded by the referencepolynucleotide. Thus, for example, an isolated polynucleotide thatencodes a polypeptide with a given percent sequence identity to thepolypeptide of SEQ ID NO: 3 are disclosed. Percent sequence identitybetween any two polypeptides can be calculated using sequence alignmentprograms and parameters described elsewhere herein. Where any given pairof polynucleotides of the invention is evaluated by comparison of thepercent sequence identity shared by the two polypeptides they encode,the percent sequence identity between the two encoded polypeptides is atleast about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.Sequences of the invention may be variants or fragments of an exemplarypolynucleotide sequence, or they may be both a variant and a fragment ofan exemplary sequence.

“Variant” protein is intended to mean a protein derived from the nativeprotein by deletion or addition of one or more amino acids at one ormore sites in the native protein and/or substitution of one or moreamino acids at one or more sites in the native protein. Variant proteinsencompassed by the present invention are biologically active, that isthey continue to possess the desired biological activity of the nativeprotein, that is, MRP activity as described herein. Such variants mayresult from, for example, genetic polymorphism or from humanmanipulation. Biologically active variants of a native MRP protein ofthe invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to the amino acid sequence for the nativeprotein as determined by sequence alignment programs and parametersdescribed elsewhere herein. A biologically active variant of a proteinof the invention may differ from that protein by as few as 1-15 aminoacid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4,3, 2, or even 1 amino acid residue. Sequences of the invention may bevariants or fragments of an exemplary protein sequence, or they may beboth a variant and a fragment of an exemplary sequence.

The proteins of the invention may be altered in various ways includingamino acid substitutions, deletions, truncations, and insertions.Methods for such manipulations are generally known in the art. Forexample, amino acid sequence variants and fragments of the MRP proteinscan be prepared by the creation of mutations in the DNA. Methods formutagenesis and nucleotide sequence alterations are well known in theart. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154: 367-382; U.S.Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques inMolecular Biology (MacMillan Publishing Company, New York) and thereferences cited therein. Guidance as to appropriate amino acidsubstitutions that do not affect biological activity of the protein ofinterest may be found in the model of Dayhoff et al. (1978) Atlas ofProtein Sequence and Structure (Nat'l. Biomed. Res. Found., Washington,D.C.), herein incorporated by reference. Conservative substitutions,such as exchanging one amino acid with another having similarproperties, may be made.

Thus, the genes and nucleotide sequences of the invention include boththe naturally occurring sequences as well as mutant forms. Likewise, theproteins of the invention encompass both naturally occurring proteins aswell as variations and modified forms thereof. Such variants willcontinue to possess the desired MRP activity. Obviously, the mutationsthat will be made in the DNA encoding the variant must not place thesequence out of reading frame and preferably will not createcomplementary regions that could produce secondary mRNA structure. See,EP Patent Application Publication No. 75,444.

The deletions, insertions, and substitutions of the protein sequencesencompassed herein are not expected to produce radical changes in thecharacteristics of the protein. However, when it is difficult to predictthe exact effect of the substitution, deletion, or insertion in advanceof doing so, one skilled in the art will appreciate that the effect willbe evaluated by routine screening assays. That is, the activity can beevaluated by the methods used in Example 1 and references cited thereinas well as by other assays known in the art.

Variant polynucleotides and proteins also encompass sequences andproteins derived from a mutagenic and recombinogenic procedure such asDNA shuffling. With such a procedure, one or more different MRP codingsequences can be manipulated to create a new MRP possessing the desiredproperties. In this manner, libraries of recombinant polynucleotides aregenerated from a population of related sequence polynucleotidescomprising sequence regions that have substantial sequence identity andcan be homologously recombined in vitro or in vivo. For example, usingthis approach, sequence motifs encoding a domain of interest may beshuffled between the MRP gene of the invention and other known MRP genesto obtain a new gene coding for a protein with an improved property ofinterest, such as an increased K_(m) in the case of an enzyme.Strategies for such DNA shuffling are known in the art. See, forexample, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91: 10747-10751;Stemmer (1994) Nature 370: 389-391; Crameri et al. (1997) NatureBiotech. 15: 436-438; Moore et al. (1997) J. Mol. Biol. 272: 336-347;Zhang et al. (1997) Proc. Natl. Acad. Sci. USA 94: 4504-4509; Crameri etal. (1998) Nature 391: 288-291; and U.S. Pat. Nos. 5,605,793 and5,837,458.

The present invention further provides a method for modulating (i.e.,increasing or decreasing) the concentration or composition of thepolypeptides of the claimed invention in a plant or part thereof.Modulation can be effected by increasing or decreasing the concentrationand/or the composition (i.e., the ratio of the polypeptides of theclaimed invention) in a plant.

In some embodiments, the method comprises transforming a plant cell witha cassette comprising a polynucleotide of the invention to obtain atransformed plant cell, growing the transformed plant cell underconditions allowing expression of the polynucleotide in the plant cellin an amount sufficient to modulate concentration and/or composition ofthe corresponding protein in the plant cell. In some embodiments, themethod comprises utilizing the polynucleotides of the invention tocreate a deletion or inactivation of the native gene. Thus, a deletionmay constitute a functional deletion, i.e., the creation of a “null”mutant, or it may constitute removal of part or all of the coding regionof the native gene. Methods for creating null mutants are well-known inthe art and include, for example, chimeraplasty as discussed elsewhereherein.

In some embodiments, the content and/or composition of polypeptides ofthe present invention in a plant may be modulated by altering, in vivoor in vitro, the promoter of a non-isolated gene of the presentinvention to up- or down-regulate gene expression. In some embodiments,the coding regions of native genes of the present invention can bealtered via substitution, addition, insertion, or deletion to decreaseactivity of the encoded enzyme. See, e.g., Kmiec, U.S. Pat. No.5,565,350; Zarling et al., PCT/US93/03868. One method of down-regulationof the protein involves using PEST sequences that provide a target fordegradation of the protein.

In addition to sense and antisense suppression, catalytic RNA moleculesor ribozymes can also be used to inhibit expression of plant genes. Theinclusion of ribozyme sequences within antisense RNAs confersRNA-cleaving activity upon them, thereby increasing the activity of theconstructs. The design and use of target RNA-specific ribozymes isdescribed in Haseloff et al. (1988) Nature 334: 585-591.

A variety of cross-linking agents, alkylating agents andradical-generating species as pendant groups on polynucleotides of thepresent invention can be used to bind, label, detect, and/or cleavenucleic acids. For example, Vlassov et al. (1986) Nucl. Acids Res. 14:4065-4076 describes covalent bonding of a single-stranded DNA fragmentwith alkylating derivatives of nucleotides complementary to targetsequences. Similar work is reported in Knorre et al. (1985) Biochimie67: 785-789. Others have also showed sequence-specific cleavage ofsingle-stranded DNA mediated by incorporation of a modified nucleotidewhich was capable of activating cleavage (Iverson and Dervan (1987) J.Am. Chem. Soc. 109: 1241-1243). Meyer et al. ((1989) J. Am. Chem. Soc.111: 8517-8519) demonstrated covalent crosslinking to a targetnucleotide using an alkylating agent complementary to thesingle-stranded target nucleotide sequence. Lee et al. ((1988)Biochemistry 27: 3197-3203) disclosed a photoactivated crosslinking tosingle-stranded oligonucleotides mediated by psoralen. Home et al.((1990) J. Am Chem. Soc. 112: 2435-2437) used crosslinking withtriple-helix-forming probes. Webb and Matteucci ((1986) J. Am. Chem.Soc. 108: 2764-2765) and Feteritz et al. ((1991) J. Am. Chem. Soc. 113:4000) used N4, N4-ethanocytosine as an alkylating agent to crosslink tosingle-stranded oligonucleotides. In addition, various compounds tobind, detect, label, and/or cleave nucleic acids are known in the art.See, for example, U.S. Pat. Nos. 5,543,507; 5,672,593; 5,484,908;5,256,648; and 5,681,941. Such embodiments are collectively referred toherein as “chemical destruction.”

In some embodiments, an isolated nucleic acid (e.g., a vector)comprising a promoter sequence is transfected into a plant cell.Subsequently, a plant cell comprising the promoter operably linked to anucleic acid or polynucleotide comprising a nucleotide sequence of thepresent invention is selected for by means known to those of skill inthe art such as, but not limited to, Southern blot, DNA sequencing, orPCR analysis using primers specific to the promoter and to the gene anddetecting amplicons produced therefrom. A plant or plant part altered ormodified by the foregoing embodiments is grown under plant-formingconditions for a time sufficient to modulate the concentration and/orcomposition of polypeptides of the present invention in the plant.Plant-forming conditions are well known in the art.

In general, when an endogenous polypeptide is modulated using themethods of the invention, the content of the polypeptide in a plant orpart or cell thereof is increased or decreased by at least 5%, 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, or more relative to a native controlplant, plant part, or cell lacking the aforementioned cassette.Modulation in the present invention may occur during and/or subsequentto growth of the plant to the desired stage of development. Modulatingnucleic acid expression temporally and/or in particular tissues can becontrolled by employing the appropriate promoter operably linked to apolynucleotide of the present invention in, for example, sense orantisense orientation.

A transformed plant or transformed plant cell of the invention is one inwhich genetic alteration, such as transformation, has been effected asto a gene of interest, or is a plant or plant cell which is descendedfrom a plant or cell so altered and which comprises the alteration. A“control” or “control plant” or “control plant cell” provides areference point for measuring changes in phenotype of the subject plantor plant cell. A control plant or plant cell may comprise, for example:(a) a wild-type plant or cell, i.e., of the same genotype as thestarting material for the genetic alteration which resulted in thesubject plant or cell; (b) a plant or plant cell of the same genotype asthe starting material but which has been transformed with a nullconstruct (i.e., with a construct which has no known effect on the traitof interest, such as a construct comprising a marker gene); (c) a plantor plant cell which is a non-transformed segregant among progeny of asubject plant or plant cell; (d) a plant or plant cell geneticallyidentical to the subject plant or plant cell but which is not exposed toconditions or stimuli that would induce expression of the gene ofinterest; or (e) the subject plant or plant cell itself, underconditions in which the gene of interest is not expressed.

The polynucleotides of the invention can be used to isolatecorresponding sequences from other organisms, particularly other plants.In this manner, methods such as PCR, hybridization, and the like can beused to identify such sequences based on their sequence homology to thesequences set forth herein. Sequences isolated based on their sequenceidentity to the entire MRP sequences set forth herein or to variants andfragments thereof are encompassed by the present invention. Suchsequences include sequences that are orthologs of the disclosedsequences. “Orthologs” is intended to mean genes derived from a commonancestral gene and which are found in different species as a result ofspeciation. Genes found in different species are considered orthologswhen their nucleotide sequences and/or their encoded protein sequencesshare at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or greater sequence identity. Functions of orthologsare often highly conserved among species. Thus, isolated sequences thatencode an MRP protein or have Lpa1 promoter activity and which hybridizeunder stringent conditions to the Lpa1 sequences disclosed herein, or tovariants or fragments thereof, are encompassed by the present invention.

In a PCR approach, oligonucleotide primers can be designed for use inPCR reactions to amplify corresponding DNA sequences from cDNA orgenomic DNA extracted from any plant of interest. Methods for designingPCR primers and PCR cloning are generally known in the art and aredisclosed in Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods andApplications (Academic Press, New York); Innis and Gelfand, eds. (1995)PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds.(1999) PCR Methods Manual (Academic Press, New York). Known methods ofPCR include, but are not limited to, methods using paired primers,nested primers, single specific primers, degenerate primers,gene-specific primers, vector-specific primers, partially-mismatchedprimers, and the like.

In hybridization techniques, all or part of a known polynucleotide isused as a probe that selectively hybridizes to other nucleic acidscomprising corresponding nucleotide sequences present in a population ofcloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNAlibraries) from a chosen organism. The hybridization probes may begenomic DNA fragments, cDNA fragments, RNA fragments, or otheroligonucleotides, and may be labeled with a detectable group such as³²P, or any other detectable marker. Thus, for example, probes forhybridization can be made by labeling synthetic oligonucleotides basedon the MRP sequences of the invention. Methods for preparation of probesfor hybridization and for construction of cDNA and genomic libraries aregenerally known in the art and are disclosed in Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.).

For example, the entire MRP sequences disclosed herein, or one or moreportions thereof, may be used as probes capable of specificallyhybridizing to corresponding MRP sequences and messenger RNAs. Toachieve specific hybridization under a variety of conditions, suchprobes include sequences that are unique among MRP sequences and are atleast about 10, 12, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35,40, 50, 60, 70, 80, 90, or more nucleotides in length. Such probes maybe used to amplify corresponding MRP sequences from a chosen plant byPCR. This technique may be used to isolate additional coding sequencesfrom a desired plant or as a diagnostic assay to determine the presenceof coding sequences in a plant. Hybridization techniques includehybridization screening of plated DNA libraries (either plaques orcolonies; see, for example, Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.).

Hybridization of such sequences may be carried out under stringentconditions. By “stringent conditions” or “stringent hybridizationconditions” is intended conditions under which a probe will hybridize toits target sequence to a detectably greater degree than to othersequences (e.g., at least 2-fold over background). Stringent conditionsare sequence-dependent and will be different in different circumstances.By controlling the stringency of the hybridization and/or washingconditions, target sequences that are 100% complementary to the probecan be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of similarity are detected (heterologous probing).Generally, a probe is less than about 1000 or 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffersmay comprise about 0.1% to about 1% SDS. Duration of hybridization isgenerally less than about 24 hours, usually about 4, 8, or 12 hours.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284:T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, “% form” is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization, and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with ≧90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermalmelting point (T_(m)); moderately stringent conditions can utilize ahybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than thethermal melting point (T_(m)); low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe thermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution), it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen (1993)Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, N.Y.); and Ausubel et al., eds. (1995) Current Protocols inMolecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience,New York). See Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).The duration of the wash time will be at least a length of timesufficient to reach equilibrium, for example, 4 hours, 8 hours, or 12hours.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, and (d)“percentage of sequence identity.”

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. Generally, the comparison window is at least 20 contiguousnucleotides in length, and optionally can be 30, 40, 50, or 100nucleotides in length, or longer. Those of skill in the art understandthat to avoid a high similarity to a reference sequence due to inclusionof gaps in the polynucleotide sequence a gap penalty is typicallyintroduced and is subtracted from the number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent sequence identity between anytwo sequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988) CABIOS 4: 11-17; the local alignmentalgorithm of Smith et al. (1981) Adv. Appl. Math. 2: 482; the globalalignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; the search-for-local-alignment-method of Pearson and Lipman(1988) Proc. Natl. Acad. Sci. 85: 2444-2448; the algorithm of Karlin andAltschul (1990) Proc. Natl. Acad. Sci. USA 87: 2264, modified as inKarlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the GCG Wisconsin Genetics Software Package, Version 10(available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.(1988) Gene 73: 237-244 (1988); Higgins et al. (1989) CABIOS 5: 151-153;Corpet et al. (1988) Nucleic Acids Res. 16: 10881-90; Huang et al.(1992) CABIOS 8: 155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331. The ALIGN program is based on the algorithm of Myers and Miller(1988) supra. A PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used with the ALIGN program when comparingamino acid sequences. The BLAST programs of Altschul et at (1990) J.Mol. Biol. 215: 403 are based on the algorithm of Karlin and Altschul(1990) supra. BLAST nucleotide searches can be performed with the BLASTNprogram, score=100, wordlength=12, to obtain nucleotide sequenceshomologous to a nucleotide sequence encoding a protein of the invention.BLAST protein searches can be performed with the BLASTX program,score=50, wordlength=3, to obtain amino acid sequences homologous to aprotein or polypeptide of the invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25: 3389.Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform aniterated search that detects distant relationships between molecules.See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST,PSI-BLAST, the default parameters of the respective programs (e.g.,BLASTN for nucleotide sequences, BLASTX for proteins) can be used. Seehttp://www.ncbi.nlm.nih.gov. Alignment may also be performed manually byinspection.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3 and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using GAP Weight of 8 and Length Weight of 2; and theBLOSUM62 scoring matrix or any equivalent program thereof. By“equivalent program” is intended any sequence comparison program that,for any two sequences in question, generates an alignment havingidentical nucleotide or amino acid residue matches and an identicalpercent sequence identity when compared to the corresponding alignmentgenerated by GAP Version 10.

GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453, to find the alignment of two complete sequences that maximizesthe number of matches and minimizes the number of gaps. GAP considersall possible alignments and gap positions and creates the alignment withthe largest number of matched bases and the fewest gaps. It allows forthe provision of a gap creation penalty and a gap extension penalty inunits of matched bases. GAP must make a profit of gap creation penaltynumber of matches for each gap it inserts. If a gap extension penaltygreater than zero is chosen, GAP must, in addition, make a profit foreach gap inserted of the length of the gap times the gap extensionpenalty. Default gap creation penalty values and gap extension penaltyvalues in Version 10 of the GCG Wisconsin Genetics Software Package forprotein sequences are 8 and 2, respectively. For nucleotide sequencesthe default gap creation penalty is 50 while the default gap extensionpenalty is 3. The gap creation and gap extension penalties can beexpressed as an integer selected from the group of integers consistingof from 0 to 200. Thus, for example, the gap creation and gap extensionpenalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity, and Similarity. The Quality is the metric maximized in orderto align the sequences. Ratio is the quality divided by the number ofbases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the GCG Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad.Sci. USA 89:10915).

(c) As used herein, “sequence identity” or “identity” in the context oftwo nucleic acid or polypeptide sequences makes reference to theresidues in the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

The use of the term “polynucleotide” is not intended to limit thepresent invention to polynucleotides comprising DNA. Those of ordinaryskill in the art will recognize that polynucleotides can compriseribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides. Such deoxyribonucleotides and ribonucleotidesinclude both naturally occurring molecules and synthetic analogues. Thepolynucleotides of the invention also encompass all forms of sequencesincluding, but not limited to, single-stranded forms, double-strandedforms, hairpins, stem-and-loop structures, and the like.

The MRP polynucleotide of the invention can be provided in expressioncassettes for expression in the plant of interest. The cassette willinclude any necessary 5′ and 3′ regulatory sequences operably linked toan MRP polynucleotide of the invention. “Operably linked” is intended tomean a functional linkage between two or more elements. For example, anoperable linkage between a polynucleotide of interest and a regulatorysequence (i.e., a promoter) is a functional link that allows forexpression of the polynucleotide of interest. Operably linked elementsmay be contiguous or non-contiguous. When used to refer to the joiningof two protein coding regions, “operably linked” is intended to meanthat the coding regions are in the same reading frame. The cassette mayadditionally contain at least one additional gene to be cotransformedinto the organism. Alternatively, the additional gene(s) can be providedon multiple expression cassettes. Such an expression cassette isprovided with a plurality of restriction sites and/or recombinationsites for insertion of the MRP polynucleotide to be under thetranscriptional regulation of the regulatory regions. The expressioncassette may additionally contain selectable marker genes. If proteinexpression is desired, the cassette may be referred to as a proteinexpression cassette and will include in the 5′-3′ direction oftranscription: a transcriptional and translational initiation region(i.e., a promoter), an MRP nucleotide sequence of the invention, and atranscriptional and translational termination region (i.e., terminationregion) functional in plants.

The regulatory regions (i.e., promoters, transcriptional regulatoryregions, and translational termination regions) and/or the MRPpolynucleotide of the invention may be native/analogous to the host cellor to each other. Alternatively, the regulatory regions and/or the MRPpolynucleotide of the invention may be heterologous to the host cell orto each other. As used herein, “heterologous” in reference to a sequenceis a sequence that originates from a foreign species, or, if from thesame species, is substantially modified from its native form incomposition and/or genomic locus by deliberate human intervention. Forexample, a promoter operably linked to a heterologous polynucleotide isfrom a species different from that from which the polynucleotide wasderived, or, if from the same/analogous species, one or both aresubstantially modified from their original form, or the promoter is notthe native promoter for the operably linked polynucleotide.

While it may be optimal to express the sequences using heterologouspromoters, the native promoter sequences (e.g., the promoter sequenceset forth in SEQ ID NO: 1) may be used. Such constructs can changeexpression levels of MRP in the plant or plant cell. Thus, the phenotypeof the plant or plant cell can be altered. The promoter sequence setforth in SEQ ID NO:1 contains a putative TATA box from nucleotides 2464to 2470; the 5′ UTR may contain an intron.

In an expression cassette, the termination region may be native with thetranscriptional initiation region, may be native with the operablylinked nucleotide sequence of interest, may be native with the planthost, or may be derived from another source (i.e., foreign orheterologous to the promoter, the nucleotide sequence of interest, theplant host, or any combination thereof). Convenient termination regionsare available from the Ti-plasmid of A. tumefaciens, such as theoctopine synthase and nopaline synthase termination regions. See alsoGuerineau et al. (1991) Mol. Gen. Genet. 262: 141-144; Proudfoot (1991)Cell 64: 671-674; Sanfacon et al. (1991) Genes Dev. 5: 141-149; Mogen etal. (1990) Plant Cell 2: 1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17: 7891-7903; andJoshi et al. (1987) Nucleic Acid Res. 15: 9627-9639.

Where appropriate, the polynucleotides may be optimized for increasedexpression in the transformed plant. That is, the genes can besynthesized using plant-preferred codons for improved expression. See,for example, Campbell and Gowri (1990) Plant Physiol. 92: 1-11 for adiscussion of host-preferred codon usage. Methods are available in theart for synthesizing plant-preferred genes. See, for example, U.S. Pat.Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic AcidsRes. 17: 477-498, herein incorporated by reference.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequencesthat may be deleterious to gene expression. The G-C content of thesequence may be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell, andthe sequence may be modified to avoid predicted hairpin secondary mRNAstructures.

The expression cassettes may additionally contain 5′ leader sequences inthe cassette construct. Such leader sequences can act to enhancetranslation. Translation leaders are known in the art and include:picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5′noncoding region) (Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA86: 6126-6130); potyvirus leaders, for example, TEV leader (Tobacco EtchVirus) (Gallie et al. (1995) Gene 165(2): 233-238), MDMV leader (MaizeDwarf Mosaic Virus) (Virology 154: 9-20), and human immunoglobulinheavy-chain binding protein (BiP) (Macejak et al. (1991) Nature 353:90-94); untranslated leader from the coat protein mRNA of alfalfa mosaicvirus (AMV RNA 4) (Jobling et al. (1987) Nature 325: 622-625); tobaccomosaic virus leader (TMV) (Gallie et al. (1989) in Molecular Biology ofRNA, ed. Cech (Liss, N.Y.), pp. 237-256); and maize chlorotic mottlevirus leader (MCMV) (Lommel et al. (1991) Virology 81: 382-385). Seealso, Della-Cioppa et al. (1987) Plant Physiol. 84: 965-968.

The expression cassette can also comprise a selectable marker gene forthe selection of transformed cells. Selectable marker genes are utilizedfor the selection of transformed cells or tissues. Marker genes includegenes encoding antibiotic resistance, such as those encoding neomycinphosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), aswell as genes conferring resistance to herbicidal compounds, such asglufosinate ammonium, bromoxynil, imidazolinones, and2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markersinclude phenotypic markers such as β-galactosidase and fluorescentproteins such as green fluorescent protein (GFP) (Su et al. (2004)Biotechnol Bioeng 85: 610-9 and Fetter et al. (2004) Plant Cell 16:215-28), cyan florescent protein (CYP) (Bolte et al. (2004) J. CellScience 117: 943-54 and Kato et al. (2002) Plant Physiol 129: 913-42),and yellow florescent protein (PhiYFP™ from Evrogen; see Bolte et al.(2004) J. Cell Science 117: 943-54).

See generally, Yarranton (1992) Curr. Opin. Biotech. 3: 506-511;Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA 89: 6314-6318;Yao et al. (1992) Cell 71: 63-72; Reznikoff (1992) Mol. Microbiol. 6:2419-2422; Barkley et al. (1980) in The Operon, pp. 177-220; Hu et al.(1987) Cell 48: 555-566; Brown et al. (1987) Cell 49: 603-612; Figge etal. (1988) Cell 52: 713-722; Deuschle et al. (1989) Proc. Natl. Acad.Aci. USA 86: 5400-5404; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA86: 2549-2553; Deuschle et al. (1990) Science 248: 480-483; Gossen(1993) Ph.D. Thesis, University of Heidelberg; Reines et al. (1993)Proc. Natl. Acad. Sci. USA 90: 1917-1921; Labow et al. (1990) Mol. Cell.Biol. 10: 3343-3356; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA89: 3952-3956; Baim et al. (1991) Proc. Natl. Acad. Sci. USA 88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19: 4647-4653;Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10: 143-162; Degenkolbet al. (1991) Antimicrob. Agents Chemother. 35: 1591-1595; Kleinschnidtet al. (1988) Biochemistry 27: 1094-1104; Bonin (1993) Ph.D. Thesis,University of Heidelberg; Gossen et al. (1992) Proc. Natl. Acad. Sci.USA 89: 5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother. 36:913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology,Vol. 78 (Springer-Verlag, Berlin); Gill et al. (1988) Nature 334:721-724. Such disclosures are herein incorporated by reference.

The above list of selectable marker genes is not meant to be limiting.Any suitable selectable marker gene can be used in the presentinvention, and one of skill in the art will be able to determine whichselectable marker gene is suitable for a particular application.

In preparing the cassette, the various DNA fragments may be manipulated,so as to provide for the DNA sequences in the proper orientation and, asappropriate, in the proper reading frame. Toward this end, adapters orlinkers may be employed to join the DNA fragments or other manipulationsmay be involved to provide for convenient restriction sites, removal ofsuperfluous DNA, removal of restriction sites, or the like. For thispurpose, in vitro mutagenesis, primer repair, restriction, annealing,resubstitutions, e.g., transitions and transversions, may be involved.

A number of promoters can be used in the practice of the invention. Thepromoters can be selected based on the desired outcome. The nucleicacids can be combined with constitutive, tissue-preferred, or otherpromoters. Such constitutive promoters include, for example, the corepromoter of the Rsyn7 promoter and other constitutive promotersdisclosed in WO 99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35Spromoter (Odell et al. (1985) Nature 313: 810-812); rice actin (McElroyet al. (1990) Plant Cell 2: 163-171); ubiquitin (Christensen et al.(1989) Plant Mol. Biol. 12: 619-632 and Christensen et al. (1992) PlantMol. Biol. 18: 675-689); pEMU (Last et al. (1991) Theon. Appl. Genet.81: 581-588); MAS (Velten et al. (1984) EMBO J. 3: 2723-2730); ALSpromoter (U.S. Pat. No. 5,659,026), and the like. Other constitutivepromoters include, for example, U.S. Pat. Nos. 5,608,149; 5,608,144;5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and6,177,611.

Chemical-regulated promoters can be used to modulate the transcriptionand/or expression of a particular nucleotide sequence in a plant throughthe application of an exogenous chemical regulator. Depending upon theobjective, the promoter may be a chemical-inducible promoter, whereapplication of the chemical induces gene expression, or achemical-repressible promoter, where application of the chemicalrepresses gene expression. Chemical-inducible promoters are known in theart and include, but are not limited to, the maize In2-2 promoter, whichis activated by benzenesulfonamide herbicide safeners, the maize GSTpromoter, which is activated by hydrophobic electrophilic compounds thatare used as pre-emergent herbicides, and the tobacco PR-1a promoter,which is activated by salicylic acid. Other chemical-regulated promotersof interest include steroid-responsive promoters (see, for example, theglucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl.Acad. Sci. USA 88: 10421-10425 and McNellis et al. (1998) Plant J.14(2): 247-257) and tetracycline-inducible and tetracycline-repressiblepromoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat. Nos. 5,814,618 and 5,789,156), hereinincorporated by reference.

Tissue-preferred promoters can be utilized to target enhanced MRPtranscription and/or expression within a particular plant tissue.Tissue-preferred promoters include those described in Yamamoto et al.(1997) Plant J. 12(2): 255-265; Kawamata et al. (1997) Plant CellPhysiol. 38(7): 792-803; Hansen et al. (1997) Mol. Gen Genet.254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2): 157-168;Rinehart et al. (1996) Plant Physiol. 112(3): 1331-1341; Van Camp et al.(1996) Plant Physiol. 112(2): 525-535; Canevascini et al. (1996) PlantPhysiol. 112(2): 513-524; Yamamoto et al. (1994) Plant Cell Physiol.35(5): 773-778; Lam (1994) Results Probl. Cell Differ. 20: 181-196;Orozco et al. (1993) Plant Mol Biol. 23(6): 1129-1138; Matsuoka et al.(1993) Proc Natl. Acad. Sci. USA 90(20): 9586-9590; and Guevara-Garciaet al. (1993) Plant J. 4(3): 495-505. Such promoters can be modified, ifnecessary, for weak expression.

Leaf-preferred promoters are known in the art. See, for example,Yamamoto et al. (1997) Plant J. 12(2): 255-265; Kwon et al. (1994) PlantPhysiol. 105: 357-67; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Gotor et al. (1993) Plant J. 3: 509-18; Orozco et al. (1993)Plant Mol. Biol. 23(6): 1129-1138; and Matsuoka et al. (1993) Proc.Natl. Acad. Sci. USA 90(20): 9586-9590.

Root-preferred promoters are known and can be selected from the manyavailable from the literature or isolated de novo from variouscompatible species. See, for example, Hire et al. (1992) Plant Mol.Biol. 20(2): 207-218 (soybean root-specific glutamine synthetase gene);Keller and Baumgartner (1991) Plant Cell 3(10): 1051-1061 (root-specificcontrol element in the GRP 1.8 gene of French bean); Sanger et al.(1990) Plant Mol. Biol. 14(3): 433-443 (root-specific promoter of themannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao etal. (1991) Plant Cell 3(1): 11-22 (full-length cDNA clone encodingcytosolic glutamine synthetase (GS), which is expressed in roots androot nodules of soybean). See also Bogusz et al. (1990) Plant Cell 2(7):633-641, where two root-specific promoters isolated from hemoglobingenes from the nitrogen-fixing nonlegume Parasponia andersonii and therelated non-nitrogen-fixing nonlegume Trema tomentosa are described. Thepromoters of these genes were linked to a β-glucuronidase reporter geneand introduced into both the nonlegume Nicotiana tabacum and the legumeLotus corniculatus, and in both instances root-specific promoteractivity was preserved. Leach and Aoyagi (1991) describe their analysisof the promoters of the highly expressed rolC and rolD root-inducinggenes of Agrobacterium rhizogenes (see Plant Science (Limerick) 79(1):69-76). They concluded that enhancer and tissue-preferred DNAdeterminants are dissociated in those promoters. Teeri et al. (1989)used gene fusion to lacZ to show that the Agrobacterium T-DNA geneencoding octopine synthase is especially active in the epidermis of theroot tip and that the TR2′ gene is root specific in the intact plant andstimulated by wounding in leaf tissue, an especially desirablecombination of characteristics for use with an insecticidal orlarvicidal gene (see EMBO J. 8(2): 343-350). The TR1′ gene, fused tonptII (neomycin phosphotransferase II) showed similar characteristics.Additional root-preferred promoters include the VfENOD-GRP3 genepromoter (Kuster et al. (1995) Plant Mol. Biol. 29(4): 759-772); androlB promoter (Capana et al. (1994) Plant Mol. Biol. 25(4): 681-691. Seealso U.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252;5,401,836; 5,110,732; and 5,023,179.

“Seed-preferred” promoters include both “seed-specific” promoters (thosepromoters active during seed development such as promoters of seedstorage proteins) as well as “seed-germinating” promoters (thosepromoters active during seed germination). See Thompson et al. (1989)BioEssays 10: 108, herein incorporated by reference. Such seed-preferredpromoters include, but are not limited to, Cim1 (cytokinin-inducedmessage); cZ19B1 (maize 19 kDa zein); milps (myo-inositol-1-phosphatesynthase); oleosin; and celA (cellulose synthase) (see WO 00/11177 andU.S. Pat. No. 6,225,529, herein incorporated by reference). Gamma-zeinis a preferred endosperm-specific promoter. Globulin (Glb-1) is apreferred embryo-specific promoter. For dicots, seed-specific promotersinclude, but are not limited to, bean β-phaseolin, napin, β-conglycinin,soybean lectin, cruciferin, and the like. For monocots, seed-specificpromoters include, but are not limited to, maize 15 kDa zein, 22 kDazein, 27 kDa zein, g-zein, waxy, shrunken 1, shrunken 2, globulin 1,etc. See also WO 00/12733, where seed-preferred promoters from end1 andend2 genes are disclosed; herein incorporated by reference.

Where low level transcription or expression is desired, weak promoterswill be used. Generally, by “weak promoter” is intended a promoter thatdrives transcription and/or expression of a coding sequence at a lowlevel. By low level is intended at levels of about 1/1000 transcripts toabout 1/100,000 transcripts to about 1/500,000 transcripts.Alternatively, it is recognized that weak promoters also encompassespromoters that are expressed in only a few cells and not in others togive a total low level of transcription and/or expression. Where apromoter is expressed at unacceptably high levels, portions of thepromoter sequence can be deleted or modified to decrease transcriptionand/or expression levels.

Such weak constitutive promoters include, for example, the core promoterof the Rsyn7 promoter (WO 99/43838 and U.S. Pat. No. 6,072,050), thecore 35S CaMV promoter, and the like. Other constitutive promotersinclude, for example, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121;5,569,597; 5,466,785; 5,399,680; 5,268,463; and 5,608,142. See also,U.S. Pat. No. 6,177,611, herein incorporated by reference.

In one embodiment, the polynucleotides of interest are targeted to thechloroplast for expression. In this manner, where the nucleic acid ofinterest is not directly inserted into the chloroplast, the expressioncassette will additionally contain a nucleic acid encoding a transitpeptide to direct the gene product of interest to the chloroplasts. Suchtransit peptides are known in the art. See, for example, Von Heijne etal. (1991) Plant Mol. Biol. Rep. 9: 104-126; Clark et al. (1989) J.Biol. Chem. 264: 17544-17550; Della-Cioppa et al. (1987) Plant Physiol.84: 965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun. 196:1414-1421; and Shah et al. (1986) Science 233: 478-481.

Chloroplast targeting sequences are known in the art and include thechloroplast small subunit of ribulose-1,5-bisphosphate carboxylase(Rubisco) (de Castro Silva Filho et al. (1996) Plant Mol. Biol.30:769-780; Schnell et al. (1991) J. Biol. Chem. 266(5): 3335-3342);5-(enolpyruvyl)shikimate-3-phosphate synthase (EPSPS) (Archer et al.(1990) J. Bioenerg. Biomemb. 22(6): 789-810); tryptophan synthase (Zhaoet al. (1995) J. Biol. Chem. 270(11): 6081-6087); plastocyanin (Lawrenceet al. (1997) J. Biol. Chem. 272(33): 20357-20363); chorismate synthase(Schmidt et al. (1993) J. Biol. Chem. 268(36): 27447-27457); and thelight harvesting chlorophyll a/b binding protein (LHBP) (Lamppa et al.(1988) J. Biol. Chem. 263: 14996-14999). See also Von Heijne et al.(1991) Plant Mol. Biol. Rep. 9: 104-126; Clark et al. (1989) J. Biol.Chem. 264: 17544-17550; Della-Cioppa et al. (1987) Plant Physiol. 84:965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun. 196:1414-1421; and Shah et al. (1986) Science 233: 478-481.

Methods for transformation of chloroplasts are known in the art. See,for example, Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab and Maliga (1993) EMBO J. 12: 601-606. The method relieson particle gun delivery of DNA containing a selectable marker andtargeting of the DNA to the plastid genome through homologousrecombination. Additionally, plastid transformation can be accomplishedby transactivation of a silent plastid-borne transgene bytissue-preferred expression of a nuclear-encoded and plastid-directedRNA polymerase. Such a system has been reported in McBride et al. (1994)Proc. Natl. Acad. Sci. USA 91: 7301-7305.

The polynucleotides of interest to be targeted to the chloroplast may beoptimized for expression in the chloroplast to account for differencesin codon usage between the plant nucleus and this organelle. In thismanner, the polynucleotides of interest may be synthesized usingchloroplast-preferred codons. See, for example, U.S. Pat. No. 5,380,831,herein incorporated by reference.

In specific embodiments, the MRP sequences of the invention can beprovided to a plant using a variety of transient transformation methods.Such transient transformation methods include, but are not limited to,the introduction of the MRP protein or variants and fragments thereofdirectly into the plant or the introduction of an MRP transcript intothe plant. Such methods include, for example, microinjection or particlebombardment. See, for example, Crossway et al. (1986) Mol Gen. Genet.202: 179-185; Nomura et al. (1986) Plant Sci. 44: 53-58; Hepler et al.(1994) Proc. Natl. Acad. Sci. 91: 2176-2180 and Hush et al. (1994) TheJournal of Cell Science 107: 775-784, all of which are hereinincorporated by reference. Alternatively, the MRP polynucleotide can betransiently transformed into the plant using techniques known in theart. Such techniques include viral vector system and the precipitationof the polynucleotide in a manner that precludes subsequent release ofthe DNA. Thus, the transcription from the particle-bound DNA can occur,but the frequency with which it is released to become integrated intothe genome is greatly reduced. Such methods include the use particlescoated with polyethylimine (PEI; Sigma #P3143).

Thus, transgenic plants having low phytic acid content and high levelsof bioavailable phosphorus can be generated by reducing or inhibitingMRP gene expression in a plant. For example, the transgenic plant cancontain a transgene comprising an inverted repeat of Lpa1 thatsuppresses endogenous Lpa1 gene expression. In this manner, transgenicplants having the low phytic acid phenotype of lpa1 mutant plants can begenerated. The transgenic plant can contain an MRP suppressor sequencealone or an MRP suppressor sequence can be “stacked” with one or morepolynucleotides of interest, including, for example, one or morepolynucleotides that can affect phytic acid levels or that provideanother desirable phenotype to the transgenic plant. For example, such atransgene can be “stacked” with similar constructs involving one or moreadditional inositol phosphate kinase genes such as ITPK-5 (inositol1,3,4-trisphosphate 5/6 kinase; e.g., SEQ ID NO: 65; see also WO03/027243), IPPK (inositol polyphosphate kinase; e.g., SEQ ID NO: 64;see also WO 02/049324), and/or a myo-inositol-1 phosphate synthase gene(milps; see U.S. Pat. Nos. 6,197,561 and 6,291,224; e.g., milps-3 (SEQID NO: 25)). With such “stacked” transgenes, even greater reduction inphytic acid content of a plant can be achieved, thereby making morephosphorus bioavailable.

Thus, in certain embodiments the nucleic acid sequences of the presentinvention can be “stacked” with any combination of nucleic acids ofinterest in order to create plants with a desired phenotype. By“stacked” or “stacking” is intended that a plant of interest containsone or more nucleic acids collectively comprising multiple nucleotidesequences so that the transcription and/or expression of multiple genesare altered in the plant. For example, antisense nucleic acids of thepresent invention may be stacked with other nucleic acids which comprisea sense or antisense nucleotide sequence of at least one of ITPK-5(e.g., SEQ ID NO: 65) and/or inositol polyphosphate kinase (IPPK; e.g.,SEQ ID NO: 64), or other genes implicated in phytic acid metabolicpathways such as Lpa3 or myo-inositol kinase (see, e.g., copendingapplication entitled, “Plant Myo-Inositol Kinase Polynucleotides andMethods of Use, Appl. No. 60/573,000, filed May 20, 2004; SEQ ID NO:68); Lpa2 (see U.S. Pat. Nos. 5,689,054 and 6,111,168); myo-inositol1-phosphate synthase (milps; e.g., SEQ ID NO: 25), myo-inositolmonophosphatase (IMP) (see WO 99/05298 and U.S. application Ser. No.10/042,465, filed Jan. 9, 2002); IP2K (e.g., SEQ ID NO: 67); and thelike. The addition of such nucleic acids could enhance the reduction ofphytic acid and InsP intermediates, thereby providing a plant with morebioavailable phosphate and/or reduced phytate. The nucleic acids of thepresent invention can also be stacked with any other gene or combinationof genes to produce plants with a variety of desired trait combinations.For example, in some embodiments, a phytase gene (e.g., SEQ ID NO: 66)is stacked with an lpa1 mutant so that phytase is expressed at highlevels in the transgenic plant. Phytase genes are known in the art. See,for example, Maugenest et al. (1999) Plant Mol. Biol. 39: 503-514;Maugenest et al. (1997) Biochem. J. 322: 511-517; WO 200183763;WO200200890.

An MRP polynucleotide also can be stacked with any otherpolynucleotide(s) to produce plants having a variety of desired traitcombinations including, for example, traits desirable for animal feedsuch as high oil genes (see, e.g., U.S. Pat. No. 6,232,529, which isincorporated herein by reference); balanced amino acids (e.g.,hordothionins; see U.S. Pat. Nos. 5,990,389; 5,885,801; 5,885,802; and5,703,409, each of which is incorporated herein by reference); barleyhigh lysine (Williamson et al. (1987) Eur. J. Biochem. 165: 99-106 andWO 98/20122); high methionine proteins (Pedersen et al. (1986) J. Biol.Chem. 261: 6279; Kirihara et al. (1988) Gene 71: 359; and Musumura etal. (1989) Plant Mol. Biol. 12: 123); increased digestibility (e.g.,modified storage proteins) and thioredoxins (U.S. Pat. No. 7,009,087).

An MRP polynucleotide also can be stacked with one or morepolynucleotides encoding a desirable trait such as a polynucleotide thatconfers, for example, insect, disease or herbicide resistance (e.g.,Bacillus thuringiensis toxic proteins; U.S. Pat. Nos. 5,366,892;5,747,450; 5,737,514; 5,723,756; 5,593,881; Geiser et al. (1986) Gene48: 109); lectins (Van Damme et al. (1994) Plant Mol. Biol. 24: 825);fumonisin detoxification genes (U.S. Pat. No. 5,792,931); avirulence anddisease resistance genes (Jones et al. (1994) Science 266: 789; Martinet al. (1993) Science 262: 1432; Mindrinos et al. (1994) Cell 78: 1089);acetolactate synthase mutants that lead to herbicide resistance such asthe S4 and/or Hra mutations; inhibitors of glutamine synthase such asphosphinothricin or basta (e.g., the bar gene); and glyphosate (e.g.,the EPSPS gene and the GAT gene; see, for example, U.S. Publication No.20040082770 and WO 03/092360). Additional polynucleotides that can bestacked with a MRP polynucleotide include, for example, those encodingtraits desirable for processing or process products such as modifiedoils (e.g., fatty acid desaturase genes (U.S. Pat. No. 5,952,544; WO94/11516); modified starches (e.g., ADPG pyrophosphorylases, starchsynthases, starch branching enzymes, and starch debranching enzymes);and polymers or bioplastics (e.g., U.S. Pat. No. 5.602,321). An MRPpolynucleotide of the invention also can be stacked with one or morepolynucleotides that provide desirable agronomic traits such as malesterility (e.g., U.S. Pat. No. 5.583,210), stalk strength, floweringtime, or transformation technology traits such as cell cycle regulationor gene targeting (e.g., WO 99/61619; WO 00/17364; WO 99/25821). Otherdesirable traits that are known in the art include high oil content;increased digestibility; balanced amino acid content; and high energycontent. Such traits may refer to properties of both seed and non-seedplant tissues, or to food or feed prepared from plants or seeds havingsuch traits; such food or feed will have improved quality.

These stacked combinations can be created by any method including butnot limited to cross breeding plants by any conventional or TopCrossmethodology, or genetic transformation. In this regard, it is understoodthat transformed plants of the invention include a plant that contains asequence of the invention that was introduced into that plant viabreeding of a transformed ancestor plant. If traits are stacked bygenetically transforming the plants, the nucleic acids of interest canbe combined at any time and in any order. More generally, where anymethod requires more than one step to be performed, it is understoodthat steps may be performed in any order that accomplishes the desiredend result. For example, a transgenic plant comprising one or moredesired traits can be used as the target to introduce further traits bysubsequent transformation. The traits can be introduced simultaneouslyin a co-transformation protocol with the polynucleotides of interestprovided by any combination of cassettes suitable for transformation.For example, if two sequences will be introduced, the two sequences canbe contained in separate cassettes (trans) or contained on the sametransformation cassette (cis). Transcription and/or expression of thesequences can be driven by the same promoter or by different promoters.In certain cases, it may be desirable to introduce a cassette that willsuppress the expression of the polynucleotide of interest. This may becombined with any combination of other cassettes to generate the desiredcombination of traits in the plant. Alternatively, traits may be stackedby transforming different plants to obtain those traits; the transformedplants may then be crossed together and progeny may be selected whichcontains all of the desired traits.

Stacking may also be performed with fragments of a particular gene ornucleic acid. In such embodiments, a plants is transformed with at leastone fragment and the resulting transformed plant is crossed with anothertransformed plant; progeny of this cross may then be selected whichcontain the fragment in addition to other transgenes, including, forexample, other fragments. These fragments may then be recombined orotherwise reassembled within the progeny plant, for example, usingsite-specific recombination systems known in the art. Such stackingtechniques could be used to provide any property associated withfragments, including, for example, hairpin RNA (hpRNA) interference orintron-containing hairpin RNA (ihpRNA) interference.

It is understood that in some embodiments the nucleic acids to bestacked with MRP can also be designed to reduce or eliminate theexpression of a particular protein, as described in detail herein forMRP. Thus, the methods described herein with regard to the reduction orelimination of expression of MRP are equally applicable to other nucleicacids and nucleotide sequences of interest, such as, for example, IPPK,ITPK-5, and milps, examples of which are known in the art and which areexpected to exist in most varieties of plants. Accordingly, thedescriptions herein of MRP fragments, variants, and other nucleic acidsand nucleotide sequences apply equally to other nucleic acids andnucleotide sequences of interest such as milps (e.g., SEQ ID NO: 25),IPPK (e.g., SEQ ID NO: 64), ITPK-5 (e.g., SEQ ID NO: 65), IP2K (e.g.,SEQ ID NO:67), and Lpa3 or MIK (myo-inositol kinase; e.g., SEQ ID NO:68). For example, an antisense construct could be designed for milpscomprising a nucleotide sequence that shared 90% sequence identity tothe complement of SEQ ID NO: 25 or was at least a 19-nucleotide fragmentof the complement of SEQ ID NO: 25.

Transformation protocols as well as protocols for introducingpolypeptides or polynucleotides into plants may vary depending on thetype of plant or plant cell, i.e., monocot or dicot, targeted fortransformation. Suitable methods of introducing polypeptides orpolynucleotides into plant cells and subsequent insertion into the plantgenome include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci.USA 83: 5602-5606, Agrobacterium-mediated transformation (Townsend etal., U.S. Pat. No. 5,563,055; Zhao et al., U.S. Pat. No. 5,981,840),direct gene transfer (Paszkowski et al. (1984) EMBO J. 3: 2717-2722),and ballistic particle acceleration (see, for example, Sanford et al.,U.S. Pat. No. 4,945,050; Tomes et al., U.S. Pat. No. 5,879,918; Tomes etal., U.S. Pat. No. 5,886,244; Bidney et al., U.S. Pat. No. 5,932,782;Tomes et al. (1995) “Direct DNA Transfer into Intact Plant Cells viaMicroprojectile Bombardment,” in Plant Cell, Tissue, and Organ Culture:Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin);McCabe et al. (1988) Biotechnology 6: 923-926); and Lec1 transformation(WO 00/28058). Also see Weissinger et al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87: 671-674(soybean); McCabe et al. (1988) Bio/Technology 6: 923-926 (soybean);Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P: 175-182(soybean); Singh et al. (1998) Theor. Appl. Genet. 96: 319-324(soybean); Datta et al. (1990) Biotechnology 8: 736-740 (rice); Klein etal. (1988) Proc. Natl. Acad. Sci. USA 85: 4305-4309 (maize); Klein etal. (1988) Biotechnology 6: 559-563 (maize); Tomes, U.S. Pat. No.5,240,855; Buising et al., U.S. Pat. Nos. 5,322,783 and 5,324,646; Tomeset al. (1995) “Direct DNA Transfer into Intact Plant Cells viaMicroprojectile Bombardment,” in Plant Cell, Tissue, and Organ Culture:Fundamental Methods, ed. Gamborg (Springer-Verlag, Berlin) (maize);Klein et al. (1988) Plant Physiol. 91: 440-444 (maize); Fromm et al.(1990) Biotechnology 8: 833-839 (maize); Hooykaas-Van Slogteren et al.(1984) Nature (London) 311: 763-764; Bowen et al., U.S. Pat. No.5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA84: 5345-5349 (Liliaceae); De Wet et al. (1985) in The ExperimentalManipulation of Ovule Tissues, ed. Chapman et al. (Longman, New York),pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84: 560-566(whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany 75: 407-413(rice); Osjoda et al. (1996) Nature Biotechnology 14: 745-750 (maize viaAgrobacterium tumefaciens); all of which are herein incorporated byreference.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5: 81-84. These plants may then be grown andeither pollinated with the same transformed strain or different strains;the resulting progeny having the desired phenotypic characteristic canthen be identified. Two or more generations may be grown to ensure thatthe desired phenotypic characteristic is stably maintained and inheritedand then seeds harvested to ensure that stable transformants exhibitingthe desired phenotypic characteristic have been achieved. In thismanner, the present invention provides transformed seed (also referredto as “transgenic seed”) having a nucleotide construct of the invention,for example, a cassette of the invention, stably incorporated into theirgenome.

As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cell tissue cultures from which maize plant can beregenerated, plant calli, plant clumps, and plant cells that are intactin plants or parts of plants such as embryos, pollen, ovules, seeds,leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks,roots, root tips, anthers, and the like. Grain is intended to mean themature seed produced by commercial growers for purposes other thangrowing or reproducing the species. Progeny, variants, and mutants ofthe regenerated plants are also included within the scope of theinvention, provided that these parts comprise the introducedpolynucleotides.

The present invention may be used for transformation of any plantspecies, including, but not limited to, monocots and dicots. Examples ofplant species of interest include, but are not limited to, corn (Zeamays), Brassica spp. (e.g., B. napus, B. rapa, B. juncea), particularlythose Brassica species useful as sources of seed oil, alfalfa (Medicagosativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghumbicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetumglaucum), proso millet (Panicum miliaceum), foxtail millet (Setariaitalica), finger millet (Eleusine coracana)), sunflower (Helianthusannuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum),soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanumtuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense,Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihotesculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple(Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao),tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana),fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica),olive (Olea europaea), papaya (Carica papaya), cashew (Anacardiumoccidentale), macadamia (Macadamia integrifolia), almond (Prunusamygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.),oats, barley, vegetables, ornamentals, and conifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum.

Conifers that may be employed in practicing the present inventioninclude, for example, pines such as loblolly pine (Pinus taeda), slashpine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine(Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir(Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitkaspruce (Picea glauca); redwood (Sequoia sempervirens); true firs such assilver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedarssuch as Western red cedar (Thuja plicata) and Alaska yellow-cedar(Chamaecyparis nootkatensis). In specific embodiments, plants of thepresent invention are crop plants (for example, corn, alfalfa,sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat,millet, tobacco, etc.). In other embodiments, corn and soybean plantsare optimal, and in yet other embodiments corn plants are optimal.

Other plants of interest include grain plants that provide seeds ofinterest, oil-seed plants, and leguminous plants. Seeds of interestinclude grain seeds, such as corn, wheat, barley, rice, sorghum, rye,etc. Oil-seed plants include cotton, soybean, safflower, sunflower,Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants includebeans and peas. Beans include guar, locust bean, fenugreek, soybean,garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea,etc.

The methods of the invention involve introducing a polypeptide orpolynucleotide into a plant. “Introducing” is intended to meanpresenting to the plant the polynucleotide or polypeptide in such amanner that the sequence gains access to the interior of a cell of theplant. The methods of the invention do not depend on a particular methodfor introducing a sequence into a plant, only that the polynucleotide orpolypeptides gains access to the interior of at least one cell of theplant. Methods for introducing polynucleotide or polypeptides intoplants are known in the art, including, but not limited to, stabletransformation methods, transient transformation methods, andvirus-mediated methods.

“Stable transformation” is intended to mean that the nucleotideconstruct introduced into a plant integrates into the genome of theplant and is capable of being inherited by the progeny thereof.“Transient transformation” is intended to mean that a polynucleotide isintroduced into the plant and does not integrate into the genome of theplant or that a polypeptide is introduced into a plant.

Thus, it is recognized that methods of the present invention do notdepend on the incorporation of an entire nucleotide construct into thegenome, only that the plant or cell thereof is altered as a result ofthe introduction of a nucleotide construct or polypeptide into a cell.In one embodiment of the invention, the genome may be altered followingthe introduction of a nucleotide construct into a cell. For example, thenucleotide construct, or any part thereof, may incorporate into thegenome of the plant. Alterations to the genome of the present inventioninclude, but are not limited to, additions, deletions, and substitutionsof nucleotides in the genome. While the methods of the present inventiondo not depend on additions, deletions, or substitutions of anyparticular number of nucleotides, it is recognized that such additions,deletions, or substitutions comprise at least one nucleotide.

In other embodiments, the polynucleotides of the invention may beintroduced into plants by contacting plants with a virus or viralnucleic acids. Generally, such methods involve incorporating anucleotide construct of the invention within a viral DNA or RNAmolecule. It is recognized that an MRP of the invention may be initiallysynthesized as part of a viral polyprotein, which later may be processedby proteolysis in vivo or in vitro to produce the desired recombinantprotein. Further, it is recognized that promoters of the invention alsoencompass promoters utilized for transcription by viral RNA polymerases.Methods for introducing nucleotide constructs into plants and expressinga protein encoded therein, involving viral DNA or RNA molecules, areknown in the art. See, for example, U.S. Pat. Nos. 5,889,191; 5,889,190;5,866,785; 5,589,367; 5,316,931, and Porta et al. (1996) MolecularBiotechnology 5: 209-221; herein incorporated by reference.

The use of the term polynucleotides herein is not intended to limit thepresent invention to nucleotide constructs comprising DNA. Those ofordinary skill in the art will recognize that nucleotide constructs,particularly polynucleotides and oligonucleotides, comprised ofribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides may also be employed in the methods disclosedherein. Thus, the nucleotide constructs of the present inventionencompass all nucleotide constructs that can be employed in the methodsof the present invention for transforming plants including, but notlimited to, those comprised of deoxyribonucleotides, ribonucleotides,and combinations thereof. Such deoxyribonucleotides and ribonucleotidesinclude both naturally occurring molecules and synthetic analogues. Thenucleotide constructs of the invention also encompass all forms ofnucleotide constructs including, but not limited to, single-strandedforms, double-stranded forms, hairpins, stem-and-loop structures, andthe like.

The promoter nucleotide sequences and methods disclosed herein areuseful in regulating expression of any heterologous nucleotide sequencein a host plant in order to vary the phenotype of a plant. Because theLpa1 promoter provides weak constitutive expression of operably linkedcoding regions, the Lpa1 promoter finds particular use in altering geneexpression in various tissues.

Various changes in phenotype are of interest including modifying thefatty acid composition in seeds, altering the amino acid content ofseeds, altering a seed's pathogen defense mechanism, and the like. Theseresults can be achieved by providing expression of heterologous productsor increased expression of endogenous products in embryos.Alternatively, the results can be achieved by providing for a reductionof expression of one or more endogenous products, particularly enzymesor cofactors in the seed. These changes result in a change in phenotypeof the transformed plant.

Genes of interest are reflective of the commercial markets and interestsof those involved in the development of the crop. Crops and markets ofinterest change, and as developing nations open up world markets, newcrops and technologies will emerge also. In addition, as ourunderstanding of agronomic traits and characteristics such as yield andheterosis increase, the choice of genes for transformation will changeaccordingly. General categories of genes of interest include, forexample, those genes involved in information, such as zinc fingers,those involved in communication, such as kinases, and those involved inhousekeeping, such as heat shock proteins. More specific categories oftransgenes, for example, include genes encoding important traits foragronomics, insect resistance, disease resistance, herbicide resistance,sterility, grain characteristics, and commercial products. Genes ofinterest include, generally, those involved in oil, starch,carbohydrate, or nutrient metabolism as well as those affecting kernelsize, sucrose loading, and the like.

Agronomically important traits such as oil, starch, and protein contentcan be genetically altered by genetic engineering in addition to usingtraditional breeding methods. Modifications include increasing contentof oleic acid, saturated and unsaturated oils, increasing levels oflysine and sulfur, providing essential amino acids, and alsomodification of starch. Hordothionin protein modifications are describedin U.S. Pat. Nos. 5,703,049, 5,885,801, 5,885,802, and 5,990,389, hereinincorporated by reference. Another example is lysine and/or sulfur richseed protein encoded by the soybean 2S albumin described in U.S. Pat.No. 5,850,016, and the chymotrypsin inhibitor from barley, described inWilliamson et al. (1987) Eur. J. Biochem. 165: 99-106, the disclosuresof which are herein incorporated by reference.

Derivatives of the coding sequences can be made by site-directedmutagenesis to increase the level of preselected amino acids in theencoded polypeptide. For example, the gene encoding the barley highlysine polypeptide (BHL) is derived from barley chymotrypsin inhibitor,U.S. application Ser. No. 08/740,682, filed Nov. 1, 1996, and WO98/20133, the disclosures of which are herein incorporated by reference.Other proteins include methionine-rich plant proteins such as fromsunflower seed (Lilley et al. (1989) Proceedings of the World Congresson Vegetable Protein Utilization in Human Foods and Animal Feedstuffs,ed. Applewhite (American Oil Chemists Society, Champaign, Ill.), pp.497-502); corn (Pedersen et al. (1986) J. Biol. Chem. 261: 6279;Kirihara et al. (1988) Gene 71: 359); and rice (Musumura et al. (1989)Plant Mol. Biol. 12: 123). Other agronomically important genes encodelatex, Floury 2, growth factors, seed storage factors, and transcriptionfactors.

Insect resistance genes may encode resistance to pests that have greatyield drag such as rootworm, cutworm, European Corn Borer, and the like.Such genes include, for example, Bacillus thuringiensis toxic proteingenes (U.S. Pat. Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756;5,593,881; and Geiser et al. (1986) Gene 48: 109, and the like.

Genes encoding disease resistance traits include detoxification genes,such as against fumonisin (U.S. Pat. No. 5,792,931); avirulence (avr)and disease resistance (R) genes (Jones et al. (1994) Science 266: 789;Martin et al. (1993) Science 262: 1432; and Mindrinos et al. (1994) Cell78: 1089); and the like.

Herbicide resistance traits may include genes coding for resistance toherbicides that act to inhibit the action of acetolactate synthase(ALS), in particular the sulfonylurea-type herbicides (e.g., theacetolactate synthase (ALS) gene containing mutations leading to suchresistance, in particular the S4 and/or Hra mutations), genes coding forresistance to herbicides that act to inhibit action of glutaminesynthase, such as phosphinothricin or basta (e.g., the bar gene), orother such genes known in the art. The bar gene encodes resistance tothe herbicide basta, the nptII gene encodes resistance to theantibiotics kanamycin and geneticin, and the ALS-gene mutants encoderesistance to the herbicide chlorsulfuron. Other genes include kinasesand those encoding compounds toxic to either male or female gametophyticdevelopment.

The quality of grain is reflected in traits such as, for example, levelsand types of oils, saturated and unsaturated, quality and quantity ofessential amino acids, and levels of cellulose. In corn, modifiedhordothionin proteins are described in U.S. Pat. Nos. 5,703,049,5,885,801, 5,885,802, and 5,990,389.

Commercial traits can also be encoded on a gene or genes that couldincrease for example, starch for ethanol production, or provideexpression of proteins. Another important commercial use of transformedplants is the production of polymers and bioplastics such as describedin U.S. Pat. No. 5,602,321. Genes such as β-Ketothiolase, PHBase(polyhydroxyburyrate synthase), and acetoacetyl-CoA reductase (seeSchubert et al. (1988) J. Bacteriol. 170: 5837-5847) facilitateexpression of polyhyroxyalkanoates (PHAs).

Exogenous products include plant enzymes and products as well as thosefrom other sources including procaryotes and other eukaryotes. Suchproducts include enzymes, cofactors, hormones, and the like. The levelof proteins, particularly modified proteins having improved amino aciddistribution to improve the nutrient value of the plant, can beincreased. This is achieved by the expression of such proteins havingenhanced amino acid content.

Some chemicals can inhibit MRP protein transport activity. For example,the sulfonylurea glibenclamide can inhibit the glucuronide transportactivity of Arabidopsis AtMRP5 and can affect its function in guardcells (Gaedeke et al. (2001) EMBO J. 20: 1875-1887; Lee et al. (2004)Plant Physiol. 134: 528-538). It is expected that glibenclamide wouldalso inhibit maize MRP3 transport activity and thus would produce a lowphytic acid phenotype.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

EXPERIMENTAL Example 1 Identification and Characterization of Maize LowPhytic Acid (Lpa) Mutant Plants

A collection of indexed mutagenized F2 families derived from several Muactive stocks (Bensen et al. (1995) Plant Cell 7: 75-84) was screenedfor seeds having high inorganic phosphate content using a rapid P_(i)assay as described below. Candidates identified as producing high-P_(i)seed were crossed with suitable maize and the progeny examined toconfirm the mutations and to determine whether the mutations wereallelic to the previously identified lpa1 mutant (referred to herein as“lpa1-1”; see U.S. Pat. No. 5,689,054; Raboy et al. (2000) PlantPhysiol. 124: 355-68). Several of these lpa lines were allelic to theearlier-identified lpa1 mutant, and these Mu-insertion alleles of thelpa1 mutant were used to clone the gene responsible for the lpa1mutation. Segregation populations were created by crossing heterozygousline PV03 57 C-05 (carrying Mu-tagged lpa1) with homozygous line GP24L3(carrying EMS allele lpa1-1). F1 plants were self-pollinated to produceF2 seeds. The phenotype of F1 plants was determined by analyzing F2 seedPi and phytic acid. Genomic DNA was extracted from leaves of individualF1 plants and used for PCR analysis as further described in Example 2.

Inorganic Phosphate (Pi) Assay

A rapid test was used to assay inorganic phosphate content in kernels.Individual kernels were placed in a 25-well plastic tray and crushed at2000 psi using a hydraulic press. Two milliliters of 1N H₂SO₄ was addedto each sample. The samples were incubated at room temperature for twohours, after which four milliliters of 0.42% ammonium molybdate-1NH₂SO₄:10% ascorbic acid (6:1) was added to each sample. Increased P_(i)content was signaled by the development of blue color within about 20minutes. Positive controls included lpa2 mutant kernels, and negativecontrols included wild-type kernels.

Determination of Phytic Acid and Inorganic Phosphate Content

Dry, mature seeds were assayed for phytic acid and P_(i) content usingmodifications of the methods described by Haug and Lantzsch ((1983) J.Sci. Food Agric. 34: 1423-1426, entitled “Sensitive method for the rapiddetermination of phytate in cereals and cereal products”) and Chen etal. ((1956) Anal. Chem. 28: 1756-1758, entitled “Microdetermination ofphosphorus”). Single kernels were ground using a Geno/Grinder2000™grinder (Sepx CertiPrep®, Metuchen, N.J.). Samples of 25 to 35 mg wereplaced into 1.5 ml Eppendorf® tubes and 1 ml of 0.4 N HCl was added tothe tubes, which were then shaken on a gyratory shaker at roomtemperature for 3.5 hours. The tubes were then centrifuged at 3,900 gfor 15 minutes. Supernatants were transferred into fresh tubes and usedfor both phytic acid and P_(i) determinations; measurements wereperformed in duplicate.

For the phytic acid assay, 35 μl of each extract was placed into wellsof a 96-well microtiter plate and then 35 μl of distilled H₂O and 140 μlof 0.02% ammonium iron (III) sulphate-0.2 N HCl were added to eachsample. The microtiter plate was covered with a rubber lid and heated ina thermal cycler at 99° C. for 30 minutes, then cooled to 4° C. and kepton an ice water bath for 15 minutes, and then left at room temperaturefor 20 minutes. The plate was then sealed with sticky foil andcentrifuged at 3,900 g at 24° C. for 30 minutes. Eighty μl of eachsupernatant was placed into wells of a fresh 96-well plate. Forabsorbance measurements, 120 μl of 1% 2,2′-bipyridine-1% thioglycolicacid solution (10 g 2,2′-bipyridine (Merck Art. 3098), 10 mlthioglycolic acid (Merck Art. 300) in ddw to 1 liter) was added to eachwell and absorbance was recorded at 519 nm using a VERSAmax™ microplatereader (Molecular Devices®, Sunnyvale, Calif.). Phytic acid content ispresented as phytic acid phosphorus (PAP). Authentic phytic acid(Sigma®, P-7660) served as a standard. This phytic acid assay alsomeasured InsP₅ and InsP₄ present in the samples.

Phytic acid was also assayed according to modifications of the methodsdescribed by Latta & Eskin (1980) (J. Agric Food Chem. 28: 1313-1315)and Vaintraub & Lapteva (1988) (Analytical Biochemistry 175: 227-230).For this assay, 25 μl of extract was placed into wells of a 96-wellmicrotiter plate; then 275 μl of a solution of 36.3 mM NaOH and 100 μlof Wade reagent (0.3% sulfosalicylic acid in 0.03% FeCl₃.6H₂O) was addedto each well. The samples were mixed and centrifuged at 39,000 g at 24°C. for 10 minutes. An aliquot of supernatant (200 μl) from each well wastransferred into a new 96-well plate, and absorbance was recorded at 500nm using a VERSAmax™ microplate reader (Molecular Devices®, Sunnyvale,Calif.).

To determine P_(i), 200 μl of each extract was placed into wells of a 96well microtiter plate. 100 μl of 30% aqueous trichloroacetic acid wasthen added to each sample and the plates were shaken and thencentrifuged at 3,900 g for 10 minutes. Fifty μl of each supernatant wastransferred into a fresh plate and 100 μl of 0.42% ammonium molybdate-1NH₂SO₄:10% ascorbic acid (7:1) was added to each sample. The plates wereincubated at 37° C. for 30 minutes and then absorbance was measured at800 nm. Potassium phosphate was used as a standard. P_(i) content waspresented as inorganic phosphate phosphorus.

Determination of Seed Myo-Inositol

Myo-inositol was quantified in dry, mature seeds and excised embryos.Tissue was ground as described above and mixed thoroughly. 100 milligramsamples were placed into 7 ml scintillation vials and 1 ml of 50%aqueous ethanol was added to each sample. The vials were then shaken ona gyratory shaker at room temperature for 1 hour. Extracts were decantedthrough a 0.45 μm nylon syringe filter attached to a 1 ml syringebarrel. Residues were re-extracted with 1 ml fresh 50% aqueous ethanoland the second extracts were filtered as before. The two filtrates werecombined in a 10×75 mm glass tube and evaporated to dryness in aSpeedVac® microcentrifuge (Savant). The myo-inositol derivative wasproduced by redissolving the residues in 50 μl of pyridine and 50 μl oftrimethylsilyl-imidazole:trimethylchlorosilane (100:1) (Tacke and Casper(1996) J. AOAC Int. 79: 472-475). Precipitate appearing at this stageindicates that the silylation reaction did not work properly. The tubeswere capped and incubated at 60° C. for 15 minutes. One milliliter of2,2,4-trimethylpentane and 0.5 milliliters of distilled water were addedto each sample. The samples were then vortexed and centrifuged at 1,000g for 5 minutes. The upper organic layers were transferred with Pasteurpipettes into 2 milliliter glass autosampler vials and crimp capped.

Myo-inositol was quantified as a hexa-trimethylsilyl ether derivativeusing an Agilent Technologies® model 5890 gas chromatograph coupled withan Agilent Technologies® model 5972 mass spectrometer. Measurements wereperformed in triplicate. One μl samples were introduced in the splitlessmode onto a 30 m×0.25 mm i.d.×0.25 μm film thickness 5MS column (AgilentTechnologies®). The initial oven temperature of 70° C. was held for 2minutes, then increased at 25° C. per minute to 170° C., then increasedat 5° C. per minute to 215° C., and finally increased at 25° C. perminute to 250° C. and then held for 5 minutes. The inlet and transferline temperatures were 250° C. Helium at a constant flow of 1 ml perminute was used as the carrier gas. Electron impact mass spectra fromm/z 50-560 were acquired at −70 eV after a 5-minute solvent delay. Themyo-inositol derivative was well resolved from other peaks in the totalion chromatograms. Authentic myo-inositol standards in aqueous solutionswere dried, derivatized, and analyzed at the same time. Regressioncoefficients of four-point calibration curves were typically0.999-1.000.

Determination of Seed Inositol Phosphates

The presence of significant amounts of inositol phosphates in matureseeds was determined by HPLC according to the Dionex Application NoteAN65, “Analysis of inositol phosphates” (Dionex Corporation®, Sunnyvale,Calif.). Tissue was ground and mixed as described above. 500 mg sampleswere placed into 20 ml scintillation vials and 5 ml of 0.4 M HCl wasadded to the samples. The samples were shaken on a gyratory shaker atroom temperature for 2 hours and then allowed to sit at 4° C. overnight.Extracts were centrifuged at 1,000 g for 10 min and filtered through a0.45 μm nylon syringe filter attached to a 5 ml syringe barrel. Justprior to HPLC analysis, 600 μl aliquots of each sample were clarified bypassage through a 0.22 μm centrifugal filter. A Dionex Corporation® DX500 HPLC with a Dionex Corporation® model AS3500 autosampler was used.25 μl samples were introduced onto a Dionex Corporation® 4×250 mmOmniPac™ PAX-100 column; Dionex Corporation® 4×50 mm OmniPac™ PAX-100guard and ATC-1 anion trap columns also were used. Inositol phosphateswere eluted at 1 ml/min with the following mobile phase gradient: 68% A(distilled water)/30% B (200 mM NaOH) for 4.0 min; 39% A/59% B at 4.1through 15.0 min; return to initial conditions at 15.1 min. The mobilephase contained 2% C (50% aqueous isopropanol) at all times to maintaincolumn performance. A Dionex Corporation® conductivity detector moduleII was used with a Dionex Corporation® ASRS-Ultra II anionself-regenerating suppressor set up in the external water mode andoperated with a current of 300 mA. Although quantitative standards wereavailable, InsP₃, InsP₄ and InsP₅ were partially but clearly resolvedfrom each other and InsP₆.

The results of the above assays demonstrated that the lpa1 mutant maizeplants have a phenotype of reduced phytic acid and increased P_(i) inseeds, but lpa1 seeds do not accumulate inositol phosphateintermediates.

Example 2 Isolation and Characterization of Maize MRP3 (Lpa1) Gene

Initially, a PCR-based method was used in an effort to clone the lpa1gene, but this effort was unsuccessful. However, a Mu-insertion site ina transcriptional activator gene was identified, and co-segregationanalysis indicated that this Mu-insertion site was very closely linkedto the Lpa1 locus. This marker, designated “TAP,” was used for map-basedcloning of the Lpa1 gene.

The PCR protocol used to identify the TAP marker is known as SAIFF:Selected Amplification of Insertion Flanking Fragments. First, genomicDNA was prepared from 5-8 plants of individual lines which were Mu⁺ andMu⁻. The genomic DNA was digested with BfaI or MseI, neither of whichcuts the Mu TIR (Terminal Inverted Repeat). The restriction endsgenerated by BfaI and MseI are the same and are compatible with theMse/Bfa adaptor.

-   -   10× RL buffer: 2.5 μl    -   BSA: 0.25 μl    -   DNA: 0.3-0.5 μg    -   Enzymes: 1 μl    -   Water: bring to 25 μl

This mixture was incubated at 37° C. for 3 to 6 hours and then denaturedat 65° C. for 20 minutes. Adaptors were then ligated to the digested DNAby adding 5 μl of adaptor mixture to each reaction:

-   -   100 mM rATP: 0.3 μl    -   10× RL buffer: 0.5 μl    -   40 uM Adaptor: 1 μl    -   T4 ligase: 1 μl (3 U/μl)    -   Water: bring to 5 μl

This mixture was then incubated at 4° C. overnight. The ligationreaction was purified with a PCR Purification Kit (Qiagen®) to removeexcess adaptors, and the reaction was brought to a final volume of 50 μlin water or elution buffer.

Control PCR was performed to check the digestion and ligation. Eitherregular Taq enzyme or another non-hot start DNA polymerase was used forthe control PCR. 1 μl of the purified ligation reaction was used as thetemplate in a 10 μl PCR reaction. The primer used was the adaptor primer(MspExt18 or the nested MseInt18 primer). DMSO was added to the mixtureto a final level of 5%. The PCR conditions were 94° C. 2 min; 35 cyclesof 94° C. 30 sec, 55° C. 30 sec, and 72° C. 2 min 30 sec; and a finalextension at 72° C. for 7 min. The reaction was then run on a 1% agarosegel and the amplification reaction visualized. Non-specificadaptor-to-adaptor amplification should occur, and there should be anice smear on the gel ranging in size from 300 bp to 3 kb.

1 μl of the purified ligation reaction was then used as the template ina 10 μl PCR reaction using Hot Start™ DNA polymerase (Qiagen®). PrimersMuExt22D and MspExt18 were added to a final concentration of 0.3-0.5 μM.DMSO was added to a final level of 5%. PCR conditions were 95° C. 15min, 20 cycles of 94° C. 30 sec, 55° C. 30 sec, and 72° C. 2 min 30 sec,followed by a final extension at 72° C. for 7 min. The reaction was thendiluted 1:10 with water.

Nested (2^(nd) round) PCR was performed with Ex Taq DNA polymerase, butany robust enzyme could be used. 1 μl of the Mu+ and Mu− pools was usedas template in a 10 μl reaction. The primers were MuInt19 and Adaptornested primers (+2 selective primers, 0.3-0.5 μM final concentration).DMSO was added to a final level of 5%. “Touchdown” PCR conditions were:95° C. 2 min, 11 cycles of 94° C. 30 sec, (65° C.-0.8° C./cycle) for 30sec, and 72° C. 2 min 30 sec, followed by 24 cycles of 94° C. 30 sec,56° C. 30 sec, and 72° C. 2 min 30 sec, with a final extension at 72′Cfor 7 min. PCR reactions were electrophoresed on a 1.5% agarose gel andexamined to identify bands which were present in the Mu+ pool but absentin the Mu− pool.

The second-round (nested) PCR was then repeated using as template firstround PCR reactions from individual plants to confirm theco-segregation. DNA fragments that were present in all Mu+ individualsand absent in all Mu− individuals were isolated from the gel andpurified. The purified DNA was cloned into a vector such as TOPO TA orpGEM-T Easy according to the manufacturer's instructions (Invitrogen™,Carlsbad, Calif.; Promega®, Madison, Wis.).

Clones were screened with PCR to identify correctly-cloned inserts foreach fragment of interest. White colonies (8) were selected andresuspended in 40 μl water; the remainder of the colony was streaked onselective media (LB+Amp) for later recovery. 1 μl of the resuspendedcolonies were used as the template in a 10 μl PCR reaction. PCRconditions were the same as described above for nested PCR, and onepositive clone was selected for each fragment.

Cultures of bacteria carrying the selected clone were grown in liquidselective media (LB+Amp). Plasmid minipreps were performed using a SpinColumn Miniprep Kit (Qiagen®). The final volume was brought to 50 μlwith elution buffer, and the minipreps were checked by digesting 2 μl ofplasmid DNA with EcoRI. The DNA was then sequenced to confirm that eachplasmid contained the MuTIR (53 bp including the MuInt19 site). Thesequence of the fragment was then used to design a fragment-specificprimer to pair with MuInt19 or MuExt22D, and co-segregation analysis wasperformed using PCR on DNA from all individuals in the segregationpopulation.

BfaI and MseI share the same adaptor:

MseI/BfaI adaptor--lower:  (SEQ ID NO: 26) 5′-TACTCAGGACTCATCGACCGT-3′MseI/BfaI adaptor--upper:  (SEQ ID NO: 27) 5′-GTGAACGGTCGATGAGTCCTGAG-3′

Adaptors were made by mixing these two oligonucleotides, denaturing at95° C. for 5 minutes, and then cooling the mixture down slowly to roomtemperature. The adaptor is designed in such a way that the originalrestriction sites are not restored after the ligation.

Adaptor Ext 18 primer (MspExtl8):  (SEQ ID NO: 28)5′-GTGAACGGTCGATGAGTC-3′ MseI/BfaI adp Int18 primer (MseInt18): (SEQ ID NO: 29) 5′-GTCGATGAGTCCTGAGTA-3′ BfaI +2 selective primers (16):(SEQ ID NO: 30) BfaIntGAA: GATGAGTCCTGAGTAGAA (SEQ ID NO: 31)BfaIntGAC: GATGAGTCCTGAGTAGAC (SEQ ID NO: 32)BfaIntGAG: GATGAGTCCTGAGTAGAG (SEQ ID NO: 33)BfaIntGAT: GATGAGTCCTGAGTAGAT (SEQ ID NO: 34)BfaIntGCA: GATGAGTCCTGAGTAGCA (SEQ ID NO: 35)BfaIntGCC: GATGAGTCCTGAGTAGCC (SEQ ID NO: 36)BfaIntGCG: GATGAGTCCTGAGTAGCG (SEQ ID NO: 37)BfaIntGCT: GATGAGTCCTGAGTAGCT (SEQ ID NO: 38)BfaIntGGA: GATGAGTCCTGAGTAGGA (SEQ ID NO: 39)BfaIntGGC: GATGAGTCCTGAGTAGGC (SEQ ID NO: 40)BfaIntGGG: GATGAGTCCTGAGTAGGG (SEQ ID NO: 41)BfaIntGGT: GATGAGTCCTGAGTAGGT (SEQ ID NO: 42)BfaIntGTA: GATGAGTCCTGAGTAGTA (SEQ ID NO: 43)BfaIntGTC: GATGAGTCCTGAGTAGTC (SEQ ID NO: 44)BfaIntGTG: GATGAGTCCTGAGTAGTG (SEQ ID NO: 45)BfaIntGTT: GATGAGTCCTGAGTAGTT MseI +2 selective primers (16):(SEQ ID NO: 46) MseIntAAA: CGATGAGTCCTGAGTAAAA (SEQ ID NO: 47)MseIntAAC: CGATGAGTCCTGAGTAAAC (SEQ ID NO: 48)MseIntAAG: CGATGAGTCCTGAGTAAAG (SEQ ID NO: 49)MseIntAAT: CGATGAGTCCTGAGTAAAT (SEQ ID NO: 50)MseIntACA: CGATGAGTCCTGAGTAACA (SEQ ID NO: 51)MseIntACC: GATGAGTCCTGAGTAACC (SEQ ID NO: 52)MseIntACG: GATGAGTCCTGAGTAACG (SEQ ID NO: 53)MseIntACT: GATGAGTCCTGAGTAACT (SEQ ID NO: 54)MseIntAGA: CGATGAGTCCTGAGTAAGA (SEQ ID NO: 55)MseIntAGC: GATGAGTCCTGAGTAAGC (SEQ ID NO: 56)MseIntAGG: GATGAGTCCTGAGTAAGG (SEQ ID NO: 57)MseIntAGT: CGATGAGTCCTGAGTAAGT (SEQ ID NO: 58)MseIntATA: CGATGAGTCCTGAGTAATA (SEQ ID NO: 59)MseIntATC: GATGAGTCCTGAGTAATC (SEQ ID NO: 60)MseIntATG: GATGAGTCCTGAGTAATG (SEQ ID NO: 61)MseIntATT: CGATGAGTCCTGAGTAATT

10× RL buffer:

-   -   100 mM Tris-HCl, pH 7.5    -   100 mM MgOAc,    -   500 mM KOAc,    -   50 mM DTT

Map-based cloning requires a high-resolution genetic map and a physicalmap around the locus of interest. Using the TAP marker, which wasclosely linked to the Lpa1 locus, the inventors identified a BAC contigcontaining about 120 BAC clones from a proprietary BAC library. PCRmarkers were developed based on BAC-end sequences and EST sequences, andthe segregating populations of individuals described above were alsoused for genetic mapping. Individual F1 seeds were phenotyped bymeasuring Pi and phytic acid content. DNA was extracted from theindividual F1 seeds with the Qiagen® Genomic DNA Purification Kit.Individuals were genotyped using PCR carried out according to theinstructions of the Expand High Fidelity PCR system (Roche®). 792individuals were analyzed to construct a fine map around the Lpa1 locus.

Based on the genetic map and the BAC physical map, the inventorsidentified two over-lapping BACs which cover the Lpa1 locus. The twoBACs, b149a.i9 and b156a.m1, were sequenced. Open reading frames in eachBAC were identified by using the Fgenesh computer program and BLASTsearching against maize EST databases. BAC b149a.i9 is 140 kb in lengthand has several ORFs predicted by Fgenesh. Only two ORFs were found tohave corresponding ESTs. One of the ORFs encodes an MRP ABC transporterprotein. Gene-specific primers were synthesized from these two ORFs andused to search for the Mu insertion in the lpa1 mutant Mu-insertionalleles. A Mu insertion was found in the MRP ABC transporter gene inlpa1 allele PV03 56 C-05. A Mu insertion was also found in the same genefor eight other lpa1 alleles. Mu is inserted in Exon 1 at nucleotide 585in Mu82978.17; at nucleotide 874 in PV03 57 C-3; and in Exon 11 atnucleotide 6069 in Mu82911.08. The remaining 6 alleles all have the sameMu insertion site as Mu82978.17. The MRP gene was also sequenced fromfour lpa1 EMS alleles. In two alleles (91286 and 94580), a stop codonwas introduced in place of codons encoding R and Q at amino acids 371and 595, respectively. In allele 91281, E was changed to L at amino acid680, while in the original lpa1-1 allele, A was mutated to V at aminoacid 1432.

The maize MRP ABC transporter gene was designated ZmMRP3 (Zea maysmultidrug resistance-associated protein 3), or Lpa1 (low phytic acid).The MRP group of the ABC transporter family includes many proteins whichare involved in diverse cellular responses. MRPs can transport a greatrange of substances. Some of the MRPs also have regulatory activity onother transporters or channel proteins. This maize MRP (ZmMRP3) is thefirst MRP shown to play a role in phytic acid metabolism and cellularfunction, and provides a new way in which phytic acid and availablephosphorus content of plant seeds may be manipulated. Previously, thephytic acid biosynthesis pathway was altered by manipulating genesencoding the enzymes that convert glucose 6-P to phytic acid. Incontrast, while the invention is not bound by a particular mechanism ofoperation, MRP is a transporter and/or transporter regulator. Thus,altering MRP expression and/or functionality in transgenic plants wouldbe expected to have minimal effects on InsP intermediates of phytic acidbiosynthesis pathway.

During the course of this study, the inventors determined that knockoutlpa1 alleles are lethal when they are homozygous. Because the Lpa1 genehas now been cloned and further characterized as disclosed herein, it isnow possible to make transgenic plants with Lpa1 expression constructsunder tight control. An advantage of using Lpa1 is that it could be usedto develop the low phytic acid trait without changing the composition ofmyo-inositol phosphate intermediates. In addition, a suppression of Lpa1expression that was limited to suppression in developing embryos couldproduce transgenic plants having low phytic acid and high availablephosphorus in seeds with minimal impact on agronomic performance.

Thus, SEQ ID NO:1 sets forth the genomic sequence of ZmMRP3 (Lpa1), SEQID NO:2 sets forth the deduced cDNA sequence, and SEQ ID NO: 3 setsforth the deduced amino acid sequence of the ZmMRP3 (Lpa1) protein. TheLpa1 protein contains 1510 amino acids and has a calculated molecularweight of about 166.8 kiloDaltons and a pI of about 8.44.

Zm-MRP3 Protein Structure

The Lpa1 polypeptide was identified as an ABC transporter, as itcontains consensus features of the ABC transporter family of proteins.ABC transporters are a large family of proteins found in bacteria,fungi, plants and animals. In coupling to ATP hydrolysis, the ABCtransporter transports a great variety of substrates across the plasmamembrane and various intracellular membranes. Among the substrates knownto be transported by ABC transporters are sugars, amino acids, inorganicacids, lipids, peptides, heavy metal ions, glutathione conjugates,alkloids, and secondary metabolites.

The member of the ABC superfamily can be divided into severalsubfamilies based on phylogenic pathways and structural features. Thenames used to define the subfamilies are historic and related to thefunction of drug resistance, although many members are not involved indrug transport. The three best characterized subfamilies are thepleiotropic drug resistance protein (PDR), multidrug resistance protein(MDR), and multidrug resistance-associated protein (MRP). The maize Lpa1is a MRP ABC transporter. Previously, two MRP genes, ZmMRP1 and ZmMRP2,have been cloned from maize and their function is not known. The Lpa1gene differs from those two ZmMRPs and thus was designated ZmMRP3.

FIG. 1A and FIG. 1B show a comparison of the Lpa1 polypeptide with Pfamconsensus sequences for the ABC transporter (“ABC_tran”; Pfam AccessionNo. PF00005; SEQ ID NO: 62) and the ABC transporter transmembrane region(“ABC_membrane”; Pfam Accession No. PF00664; SEQ ID NO: 63). All ABCproteins consist of one or two copies of a modular structure which hastwo basic structural elements: an integral transmembrane domain (TMD)and a cytosolic ATP-binding domain (also known as nucleotide bindingfold, or NBF). The NBF is involved in binding ATP and it contains aWalker A box, an ABC signature motif, and a Walker B box. The Walker Aand B boxes also are found in other nucleotide-binding proteins, such asP-, F- and V-ATPase, G-proteins and adenylate kinase. The ABC signaturemotif, however, is unique to the NBFs of ABC transporters.

The members of the MRP subfamily of ABC transporters have two copies ofthe modular structure (see FIG. 1). Maize ZmMRP3 contains about 10transmembrane spans in the first copy and 4 in the second copy. TwoATP-binding domains of ZmMRP3 are located at amino acids 631-843 andamino acids 1267-1450, respectively. Within the ATP-binding domains, aWalker A box is at amino acids 664-672 (GVIGSGKSS; SEQ ID NO: 18) andamino acids 1301-1309 (GRTGSGKST; SEQ ID NO: 19), an ABC signature motifis at amino acids 754-765 (LSGGQKQRVQLA; SEQ ID NO: 20) and amino acids1404-1415 (WSVGQRQLIALG; SEQ ID NO: 21), and a Walker B box is at aminoacids 774-779 (IYLLDD; SEQ ID NO: 22) and amino acids 1424-1428 (ILVLD;SEQ ID NO: 23). The second ATP-binding domain of ZmMRP3 is followed by aC1 domain with a motif of IAHRI (SEQ ID NO: 24) from amino acids1458-1462.

The MRP gene was amplified from different maize lines by PCR andsequenced. This revealed a variant Lpa1 polypeptide (SEQ ID NO: 5) whichdiffers from Lpa1 at positions 3, 17, and 61. This variant polypeptideis encoded by the cDNA set forth in SEQ ID NO: 4.

Example 3 Identification of Lpa1 Homologs in Other Plants

Database searches identified similar proteins from other plants whichwere not previously known to have a role in phytic acid metabolism asdiscussed herein. Accordingly, the invention additionally provides Lpa1plant proteins and proteins comprising Lpa1 consensus sequences anddomains as well as polynucleotides encoding them.

The maize MRP3 (Lpa1) gene is located on the short arm of chromosome 1and consists of 11 exons and 10 introns. It is well known that there issignificant conservation of gene content and gene order among thegenomes of the plant family Gramineae. Previously, extensive studieshave been focused on comparison of rice and maize gene linkage blocksand a comparative map established. Using the Lpa1 locus and itssurrounding sequences, the inventors found the corresponding region inrice on chromosome 3 and identified an MRP gene in this region. Althoughtwelve rice MRP genes had been annotated previously (Jasinski et al.(2003) Plant Physiol. 131: 1169-77), this annotation did not includethis MRP on chromosome 3, which we designated OsMRP13 (SEQ ID NO: 6).OsMRP13 has the same number of exons and introns as the maize Lpa1 geneZmMRP3 and encodes a protein of 1505 amino acids (SEQ ID NO: 7). Themaize MzMRP3 and rice OsMRP13 genes share 83% nucleotide sequenceidentity and the encoded proteins share 91% amino acid sequence identity(see FIGS. 4 and 5). The two genes also share similar structures (seeFIG. 2). The inventors conducted a Lynx™ study to determine theexpression patterns of the rice gene. Lynx™ gene expression profilingtechnology utilizes massively parallel signature sequence (MPSS; seeBrenner et al. (2000) Nature Biotechnology 18: 630-634; Brenner et al.(2000) Proc. Nat'l. Acad. Sci. USA 97: 1665-1670). MPSS generates 17-mersequence tags of millions of cDNA molecules, which are cloned onmicrobeads. The technique provides an unprecedented depth andsensitivity of mRNA detection, including messages expressed at very lowlevels. The Lynx™ database search revealed that the rice gene OsMRP13 isexpressed in developing seeds but has lower levels of expression inother tissues. It is very likely that the rice OsMRP13 has the samefunction as the maize Lpa1 gene in phytic acid metabolism in developingseeds.

Arabidopsis has 14 known MRP genes (AtMRP15 is a pseudogene). Theinventors discovered that AtMRP5 has the same exon/intron organizationas the maize ZmMRP3 gene, and that the sizes of corresponding exons andintrons also are similar. The maize ZmMRP3 and Arabidopsis AtMRP5 share62% nucleotide sequence identity and 67% amino acid sequence identity.Among the 14 known Arabidopsis MRPs, AtMRP5 shares the highest level ofsequence identity with ZmMRP3. A Lynx™ study was performed on AtMRP5 andconfirmed that AtMRP5 is expressed in Arabidopsis seeds. It remains tobe determined whether Arabidopsis AtMRP5 has the same function as maizeZmMRP3 in phytic acid metabolism.

A soybean homolog of maize ZmMRP3 also was identified by searching asoybean EST database. The inventors conducted a Lynx™ study tocharacterize the expression of the soybean gene (corresponding to thesequence set forth in SEQ ID NO: 10). The Lynx™ study revealed that thesoybean gene is expressed in developing seeds but has lower levels ofexpression in other tissues. A study of EST distribution in variousplant tissues also indicated that the soybean gene expression isseed-preferred.

Example 4 Stacking Lpa1 with Other Inositol Phosphate Kinase Genes

By “stacking” (i.e., transforming a plant with) constructs designed toreduce or eliminate the expression of Lpa1 and other proteins, it isexpected that the reduction of phytic acid and increase in availablephosphorus will be enhanced in comparison to plants transformed withconstructs designed to reduce or eliminate the expression of Lpa1 alone.Accordingly, expression cassettes are prepared making use of invertedrepeat constructs known as Inverted Repeats Without Terminators, or“IRNTs.” The first and second portion of such constructs self-hybridizeto produce a hairpin structure which can suppress expression of therelevant endogenous gene. Each expression cassette contains an IRNT(“Lpa1 IRNT”) that can suppress endogenous Lpa1 gene expression. ThisLpa1 IRNT includes two portions of an Lpa1 inverted repeat surroundingthe Adh1 gene intron. Other expression cassettes contain an additionalIRNT that can suppress expression of IPPK, ITPK-5, myo-inositol kinase(MIK), IP2K, phytase, and MI1PS3, respectively. “Glb1” indicates theglobulin 1 promoter, and “Ole” indicates the oleosin promoter. Eachexpression cassette is provided in a plasmid which contains additionaluseful features for transformation and expression in plants. Lpa1constructs can also be stacked with constructs designed to increase theexpression of other proteins, such as, for example, phytase.

The plasmids are inserted into Agrobacterium vectors and used totransform maize cells. Sample protocols for creation of Agrobacteriumstrains harboring a plasmid are described, for example, in Lin (1995) inMethods in Molecular Biology, ed. Nickoloff, J. A. (Humana Press,Totowa, N.J.). Successful transformation can be verified by restrictionanalysis of the plasmid after transformation back into E. coli DH5αcells. The Agrobacterium is used to transform a host plant such asmaize, and the resulting transgenic plants are screened fortransformation and for phytic acid phenotype as described in detailabove.

In some embodiments, the Lpa1 gene is mutated and the mutated Lpa1 geneis over-expressed in order to generate transgenic plants with dominantphenotype of reduced Lpa1 activity. For example, the mutation found inEMS-generated allele lpa1-1 is A1432V (i.e., the alanine at position1432 is changed to valine). This mutation can be introduced into apolynucleotide by PCR-based mutagenesis in which a primer is synthesizedwith an altered nucleotide corresponding to the desired change. Theresulting PCR product is then ligated with other fragments to make afull-length mutated Lpa1 gene carrying the lpa1-1 mutation. Atransformation construct consisting of the mutated Lpa1 gene driven bythe oleosin promoter could be used to produce transgenic plants havingthe dominant phenotype of reduced Lpa1 activity; these plants wouldyield grain with reduced phytate and increased available phosphorus.

Total knockout of the Lpa1 gene (for example, in Mutator-insertionalleles) is lethal. It is believed that the lethality of an Lpa1knockout could be rescued by overexpressing phytase in a plant lackingLpa1 activity.

Plants with Lpa1 constructs or mutations can then be crossed with plantscontaining other constructs to obtain progeny containing multipleconstructs. Thus, for example, a plant with an Lpa1 construct can becrossed with a plant containing an Lpa3 construct; progeny containingboth the Lpa1 and the Lpa3 construct may then be obtained.

Example 5 Production of Lpa1 Transgenic Plants usingAgrobacterium-Mediated Transformation

For Agrobacterium-mediated transformation of maize with an Lpa1construct of the invention, preferably the method of Zhao is employed(U.S. Pat. No. 5,981,840, and PCT patent publication WO98/32326; thecontents of which are hereby incorporated by reference). Briefly,immature embryos are isolated from maize and the embryos contacted witha suspension of Agrobacterium, where the bacteria are capable oftransferring the Lpa1 construct to at least one cell of at least one ofthe immature embryos (step 1: the infection step). In this step theimmature embryos are preferably immersed in an Agrobacterium suspensionfor the initiation of inoculation. The embryos are co-cultured for atime with the Agrobacterium (step 2: the co-cultivation step).Preferably the immature embryos are cultured on solid medium followingthe infection step. Following this co-cultivation period, an optional“resting” step is contemplated. In this resting step, the embryos areincubated in the presence of at least one antibiotic known to inhibitthe growth of Agrobacterium without the addition of a selective agentfor plant transformants (step 3: resting step). Preferably the immatureembryos are cultured on solid medium with antibiotic, but without aselecting agent, for elimination of Agrobacterium and for a restingphase for the infected cells. Next, inoculated embryos are cultured onmedium containing a selective agent and growing transformed callus isrecovered (step 4: the selection step). Preferably, the immature embryosare cultured on solid medium with a selective agent resulting in theselective growth of transformed cells. The callus is then regeneratedinto plants (step 5: the regeneration step), and preferably calli grownon selective medium are cultured on solid medium to regenerate theplants.

Bombardment and Culture Media

Bombardment medium (560Y) comprises 4.0 g/l N6 basal salts (Sigma®C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000× Sigma®-1511), 0.5 mg/lthiamine HCl, 120.0 g/l sucrose, 1.0 mg/l 2,4-D, and 2.88 g/l L-proline(brought to volume with D-I H₂O following adjustment to pH 5.8 withKOH); 2.0 g/l Gelrite™ (added after bringing to volume with D-I H₂O);and 8.5 mg/l silver nitrate (added after sterilizing the medium andcooling to room temperature). Selection medium (560R) comprises 4.0 g/lN6 basal salts (Sigma® C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000×Sigma®-1511), 0.5 mg/l thiamine HCl, 30.0 g/l sucrose, and 2.0 mg/l2,4-D (brought to volume with D-I H₂O following adjustment to pH 5.8with KOH); 3.0 g/l Gelrite™ (added after bringing to volume with D-IH₂O); and 0.85 mg/l silver nitrate and 3.0 mg/l bialaphos (both addedafter sterilizing the medium and cooling to room temperature).

Plant regeneration medium (288J) comprises 4.3 g/l MS salts (Gibco®11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g nicotinic acid,0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/l glycinebrought to volume with polished D-I H₂O) (Murashige and Skoog (1962)Physiol. Plant. 15:473), 100 mg/l myo-inositol, 0.5 mg/l zeatin, 60 g/lsucrose, and 1.0 ml/l of 0.1 mM abscisic acid (brought to volume withpolished D-I H₂O after adjusting to pH 5.6); 3.0 g/l Gelrite™ (addedafter bringing to volume with D-I H₂O); and 1.0 mg/l indoleacetic acidand 3.0 mg/l bialaphos (added after sterilizing the medium and coolingto 60° C.). Hormone-free medium (272V) comprises 4.3 g/l MS salts(Gibco® 11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g/lnicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40g/l glycine brought to volume with polished D-I H₂O), 0.1 g/lmyo-inositol, and 40.0 g/l sucrose (brought to volume with polished D-IH₂O after adjusting pH to 5.6); and 6 g/l Bacto-agar (added afterbringing to volume with polished D-I H₂O), sterilized and cooled to 60°C.

Example 6 Production of Lpa1 Transgenic Plants using Soybean EmbryoTransformation

Soybean embryos are bombarded with a plasmid containing an Lpa1construct as follows. To induce somatic embryos, cotyledons 3-5 mm inlength dissected from surface-sterilized, immature seeds of the soybeancultivar A2872 are cultured in the light or dark at 26° C. on anappropriate agar medium for six to ten weeks. Somatic embryos producingsecondary embryos are then excised and placed into a suitable liquidmedium. After repeated selection for clusters of somatic embryos thatmultiplied as early, globular-staged embryos, the suspensions aremaintained as described below.

Soybean embryogenic suspension cultures can maintained in 35 ml liquidmedia on a rotary shaker at 150 rpm at 26° C. with florescent lights ona 16:8 hour day/night schedule. Cultures are subcultured every two weeksby inoculating approximately 35 mg of tissue into 35 ml of liquidmedium.

Soybean embryogenic suspension cultures may then be transformed by themethod of particle gun bombardment (Klein et al. (1987) Nature (London)327:70-73, U.S. Pat. No. 4,945,050). A Du Pont Biolistic PDS1000/HEinstrument (helium retrofit) can be used for these transformations.

A selectable marker gene that can be used to facilitate soybeantransformation is a transgene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell et al. (1985) Nature 313: 810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al. (1983) Gene 25:179-188), and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The expression cassette comprising the Lpa1 constructoperably linked to the CaMV 35S promoter can be isolated as arestriction fragment. This fragment can then be inserted into a uniquerestriction site of the vector carrying the marker gene.

To 50 μl of a 60 mg/ml 1 μm gold particle suspension is added (inorder): 5 μl DNA (1 μg/μl), 20 μl spermidine (0.1 M), and 50 μl CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μl 70% ethanol andresuspended in 40 μl of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five microliters ofthe DNA-coated gold particles are then loaded on each macro carrierdisk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm Petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi, and the chamber is evacuated to a vacuum of 28inches mercury. The tissue is placed approximately 3.5 inches away fromthe retaining screen and bombarded three times. Following bombardment,the tissue can be divided in half and placed back into liquid andcultured as described above.

Five to seven days post bombardment, the liquid media may be exchangedwith fresh media, and eleven to twelve days post-bombardment with freshmedia containing 50 mg/ml hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post-bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 7 Production of Lpa1 Transgenic Plants using Brassica Napus SeedTransformation

Brassica napus seeds are transformed using a transformation andregeneration protocol modified from Mehra-Palta et al. (1991), “GeneticTransformation of Brassica napus and Brassica rapa,” in Proc. 8^(th)GCIRC Congr., ed. McGregor (University Extension Press, Saskatoon,Sask., Canada), pp. 1108-1115 and Stewart et al. (1996), “Rapid DNAExtraction From Plants,” in Fingerprinting Methods Based on ArbitrarilyPrimed PCR, Micheli and Bova, eds. (Springer, Berlin), pp. 25-28. SeeCardoza and Stewart (2003) Plant Cell Rep. 21: 599-604.

Seeds are surface-sterilized for 5 minutes with 10% sodium hypochloritewith 0.1% Tween™ added as a surfactant, rinsed for one minute with 95%ethanol, and then washed thoroughly with sterile distilled water. Seedsare germinated on MS basal medium (Murashige and Skoog (1962) Physiol.Plant 15: 473-497) containing 20 g/liter sucrose and 2 g/liter Gelrite™.Hypocotyls are excised from 8- to 10-day-old seedlings, cut into 1-cmpieces, and preconditioned for 72 hours on MS medium supplemented with 1mg/liter 2,4-D (2,4-dichlorophenoxy acetic acid) and containing 30g/liter sucrose and 2 g/liter Gelrite™.

Agrobacterium containing a plasmid comprising an Lpa1 construct of theinvention is grown overnight in liquid LB medium to an OD₆₀₀ of 0.8,pelleted by centrifugation, and resuspended in liquid callus inductionmedium containing acetosyringone at a final concentration of 0.05 mM.Agrobacterium is then cocultivated with the preconditioned hypocotylsegments for 48 hours on MS medium with 1 mg/liter 2,4-D. After thecocultivation period, explants are transferred to MS medium containing 1mg/liter 2,4-D, 400 mg/liter timentin, and 200 mg/liter kanamycin toselect for transformed cells. After 2 weeks, in order to promoteorganogenesis, the explants are transferred to MS medium containing 4mg/liter BAP (6-benzylaminopurine), 2 mg/liter zeatin, 5 mg/liter silvernitrate, antibiotics selective for the transformation construct, 30g/liter sucrose, and 2 g/liter Gelrite™. After an additional 2 weeks, inorder to promote shoot development, tissue is transferred to MS mediumcontaining 3 mg/liter BAP, 2 mg/liter zeatin, antibiotics, 30 g/litersucrose, and 2 g/liter Gelrite™. Shoots that develop are transferred forelongation to MS medium containing 0.05 mg/liter BAP, 30 g/litersucrose, antibiotics, and 3 g/liter Gelrite™. Elongated shoots are thentransferred to root development medium containing half-strength MSsalts, 10 mg/liter sucrose, 3 g/liter Gelrite™, 5 mg/liter IBA(indole-3-butyric acid), and antibiotics. All cultures are maintained at25° C.+/−2° C. in a 16-hour light/8-hour dark photoperiod regime withlight supplied by cool white daylight fluorescent lights. The rootedshoots are transferred to soil and grown under the same photoperiodregime at 20° C. in a plant growth chamber.

Transformation of plants with the Lpa1 construct is confirmed using PCRof DNA extracted from putative transgenic plants.

Example 8 Variants of Lpa1

A. Variant Nucleotide Sequences of Lpa1 (SEQ ID NO: 2) that do not Alterthe Encoded Amino Acid Sequence

The Lpa1 nucleotide sequence set forth in SEQ ID NO: 2 is used togenerate variant nucleotide sequences having the nucleotide sequence ofthe open reading frame with about 70%, 76%, 81%, 86%, 92%, and 97%nucleotide sequence identity when compared to the starting unaltered ORFnucleotide sequence of SEQ ID NO: 2. In some embodiments, thesefunctional variants are generated using a standard codon table. In theseembodiments, while the nucleotide sequence of the variant is altered,the amino acid sequence encoded by the open reading frame does notchange.

B. Variant Amino Acid Sequences of Lpa1

Variant amino acid sequences of Lpa1 are generated. In this example, oneamino acid is altered. Specifically, the open reading frame set forth inSEQ ID NO: 2 is reviewed to determined the appropriate amino acidalteration. The selection of the amino acid to change is made byconsulting the protein alignment (with the other homologs or orthologsand other gene family members from various species). See FIGS. 1, 4, and5. An amino acid is selected that is deemed not to be under highselection pressure (not highly conserved) and which is rather easilysubstituted by an amino acid with similar chemical characteristics(i.e., similar functional side-chain). Using the alignments set forth inFIGS. 1, 4, and 5, an appropriate amino acid can be changed. Variantshaving about 70%, 75%, 80%, 85%, 90%, 95%, and 97% nucleic acid sequenceidentity to SEQ ID NO: 2 are generated using this method.

C. Additional Variant Amino Acid Sequences of Lpa1

In this example, artificial protein sequences are created having about80%, 85%, 90%, 95%, and 97% identity relative to the reference proteinsequence. This latter effort requires identifying conserved and variableregions from the alignments set forth in FIGS. 1, 4, and 5 and then thejudicious application of an amino acid substitutions table. These partswill be discussed in more detail below.

Largely, the determination of which amino acid sequences are altered ismade based on the conserved regions among MRPs. See FIGS. 1, 4, and 5.It is recognized that conservative substitutions can be made in theconserved regions below without altering function. In addition, one ofskill will understand that functional variants of the Lpa1 sequence ofthe invention can have minor non-conserved amino acid alterations in theconserved domain.

Artificial protein sequences are then created that are different fromthe original in the intervals of 80-85%, 85-90%, 90-95%, and 95-100%identity. Midpoints of these intervals are targeted, with liberallatitude of plus or minus 1%, 2%, or 3%, for example. The amino acidssubstitutions will be effected by a custom Perl script. The substitutiontable is provided below in Table 1.

TABLE 1 Substitution Table Strongly Similar and Rank of Amino OptimalOrder to Acid Substitution Change Comment I L, V 1 50:50 substitution LI, V 2 50:50 substitution V I, L 3 50:50 substitution A G 4 G A 5 D E 6E D 7 W Y 8 Y W 9 S T 10 T S 11 K R 12 R K 13 N Q 14 Q N 15 F Y 16 M L17 First methionine cannot change H Na No good substitutes C Na No goodsubstitutes P Na No good substitutes

First, any conserved amino acids in the protein that should not bechanged is identified and “marked off” for insulation from thesubstitution. The start methionine will of course be added to this listautomatically. Next, the changes are made.

H, C, and P are not changed in any circumstance. The changes will occurwith isoleucine first, sweeping N-terminal to C-terminal, then leucine,and so on down the list until the desired target of percent change isreached. Interim number substitutions can be made so as not to causereversal of changes. The list is ordered 1-17, so start with as manyisoleucine changes as needed before leucine, and so on down tomethionine. Clearly, many amino acids will in this manner not need to bechanged. Changes between L, I, and V will involve a 50:50 substitutionof the two alternate optimal substitutions.

The variant amino acid sequences are written as output. Perl script isused to calculate the percent identities. Using this procedure, variantsof Lpa1 are generated having about 80%, 85%, 90%, and 95% amino acididentity to the starting unaltered ORF nucleotide sequence of SEQ ID NO:2.

Example 9 Pedigree Breeding

Pedigree breeding starts with the crossing of two genotypes, such as atransformed (i.e., transgenic) inbred line and one other elite inbredline having one or more desirable characteristics that is lacking orwhich complements the first transgenic inbred line. If the two originalparents do not provide all the desired characteristics, other sourcescan be included in the breeding population. In the pedigree method,superior segregating plants are selfed and selected in successive filialgenerations. In the succeeding filial generations the heterozygouscondition gives way to homogeneous lines as a result of self-pollinationand selection. Typically in the pedigree method of breeding, five ormore successive filial generations of selfing and selection arepracticed: F1→F2; F2→F3; F3→F4; F4→F5, etc. After a sufficient amount ofinbreeding, successive filial generations will serve to increase seed ofthe developed inbred. Preferably, the inbred line comprises homozygousalleles at about 95% or more of its loci.

In addition to being used to create a backcross conversion, backcrossingcan also be used in combination with pedigree breeding to modify atransgenic inbred line and a hybrid that is made using the transgenicinbred line. Backcrossing can be used to transfer one or morespecifically desirable traits from one line, the donor parent, to aninbred called the recurrent parent, which has overall good agronomiccharacteristics yet lacks that desirable trait or traits.

Therefore, an embodiment of this invention is a method of making abackcross conversion of a maize transgenic inbred line containing anLpa1 construct, comprising the steps of crossing a plant of an elitemaize inbred line with a donor plant comprising a mutant gene ortransgene conferring a desired trait, selecting an F1 progeny plantcomprising the mutant gene or transgene conferring the desired trait,and backcrossing the selected F1 progeny plant to a plant of the elitemaize inbred line. This method may further comprise the step ofobtaining a molecular marker profile of the elite maize inbred line andusing the molecular marker profile to select for a progeny plant withthe desired trait and the molecular marker profile of the maize eliteinbred line. In the same manner, this method may be used to produce anF1 hybrid seed by adding a final step of crossing the desired traitconversion of the elite maize inbred line with a different maize plantto make F1 hybrid maize seed comprising a mutant gene or transgeneconferring the desired trait.

Recurrent Selection and Mass Selection

Recurrent selection is a method used in a plant breeding program toimprove a population of plants. The method entails individual plantscross-pollinating with each other to form progeny. The progeny are grownand superior progeny are selected by any number of selection methods,which include individual plant, half-sib progeny, full-sib progeny,selfed progeny and topeross yield evaluation. The selected progeny arecross-pollinated with each other to form progeny for another population.This population is planted and again superior plants are selected tocross-pollinate with each other. Recurrent selection is a cyclicalprocess and therefore can be repeated as many times as desired. Theobjective of recurrent selection is to improve the traits of apopulation. The improved population can then be used as a source ofbreeding material to obtain inbred lines to be used in hybrids or usedas parents for a synthetic cultivar. A synthetic cultivar is theresultant progeny formed by the intercrossing of several selectedinbreds.

Mass selection is a useful technique when used in conjunction withmolecular marker enhanced selection. In mass selection seeds fromindividuals are selected based on phenotype and/or genotype. Theseselected seeds are then bulked and used to grow the next generation.Bulk selection requires growing a population of plants in a bulk plot,allowing the plants to self-pollinate, harvesting the seed in bulk andthen using a sample of the seed harvested in bulk to plant the nextgeneration. Instead of self-pollination, directed pollination could beused as part of the breeding program.

Mutation Breeding

Mutation breeding is one of many methods that could be used to introducenew traits into a particular maize inbred line. Mutations that occurspontaneously or are artificially induced can be useful sources ofvariability for a plant breeder. The goal of artificial mutagenesis isto increase the rate of mutation for a desired characteristic. Mutationrates can be increased by many different means. Such means include:temperature; long-term seed storage; tissue culture conditions;radiation such as X-rays, Gamma rays (e.g., cobalt 60 or cesium 137),neutrons, (product of nuclear fission by uranium 235 in an atomicreactor), Beta radiation (emitted from radioisotopes such as phosphorus32 or carbon 14), or ultraviolet radiation (preferably from 2500 to 2900nm); genetic means such as transposable elements or DNA damage repairmutations; chemical mutagens (such as base analogues (5-bromo-uracil);and related compounds (8-ethoxy caffeine), antibiotics (streptonigrin),alkylating agents (sulfur mustards, nitrogen mustards, epoxides,ethylenamines, sulfates, sulfonates, sulfones, lactones), azide,hydroxylamine, nitrous acid, or acridines. Once a desired trait isobserved through mutagenesis the trait may then be incorporated intoexisting germplasm by traditional breeding techniques, such asbackcrossing. Details of mutation breeding can be found in Fehr (1993)“Principals of Cultivar Development” (Macmillan Publishing Company), thedisclosure of which is incorporated herein by reference. In addition,mutations created in other lines may be used to produce a backcrossconversion of a transgenic elite line that comprises such mutation.

Example 10 Gene Silencing with the Lpa1 Promoter

The promoter of a target gene (e.g., Lpa1) is inactivated by introducinginto a plant an expression cassette comprising a promoter and aninverted repeat of fragments of the Lpa1 promoter. For example, anexpression cassette may be created that comprises the Ole promoteroperably linked to an inverted repeat comprising fragments of the Lpa1promoter that are approximately 200 bp in length and that are separatedby the Adh1 intron. The Lpa1 promoter fragments may be selected from aportion of the promoter which is rich in CpG islands, such as, forexample, the 3′ portion of the Lpa1 promoter. The sequence of the Lpa1promoter is set forth in nucleotides 1-3134 of SEQ ID NO: 1. Theexpression cassette is used to transform a plant, which is then assayedfor lack of expression of the Lpa1 gene. While the invention is notbound by any particular mechanism of operation, the method is thought toproduce a small RNA molecule which recognizes the native promoter of thetarget gene and leads to methylation and inactivation (i.e., genesilencing) of the native promoter. Consequently, the gene associatedwith the promoter is not expressed. This trait is heritable andcosegregates with the transgenic construct.

Example 11 Construction of an Lpa1 Silencing Plasmid Driven by KT13

An expression cassette was prepared making use of an inverted repeatconstruct known as Inverted Repeats Without Terminators, or “IRNTs.” Thefirst and second portion of such a construct hybridize to each other toproduce a hairpin structure which can suppress expression of thecorresponding endogenous gene (e.g., Lpa1). In this Lpa1 IRNT, the firstand second portions are separated by a “spacer” portion.

To make the spacer DNA, a polynucleotide fragment encoding part of thesoybean Fad2-1 and soybean Fad2-2 proteins (Heppard et al. (1996) PlantPhysiol. 110: 311-9) was produced as follows. First, a recombinant DNAfragment (“KSFad2-hybrid”, set forth in SEQ ID NO: 72) was produced thatcontained a polynucleotide fragment of about 890 nucleotides comprisingabout 470 nucleotides from the soybean Fad2-2 gene and about 420nucleotides from the soybean Fad2-1 gene. This KSFad2-hybrid recombinantDNA fragment was constructed by PCR amplification as follows. A DNAfragment of approximately 0.47 kb was obtained by PCR amplificationusing primers KS1 (SEQ ID NO: 73) and KS2 (SEQ ID NO: 74) from atemplate of genomic DNA purified from leaves of Glycine max cv. Jack. Anapproximately 0.42 kb DNA fragment was obtained from the same templateby PCR amplification using primers KS3 (SEQ ID NO: 75) and KS4 (SEQ IDNO: 76). The 0.47 kb DNA fragment and 0.42 kb DNA fragment weregel-purified using GeneClean® (Qbiogene, Irvine Calif.), and then weremixed together and used as a template for PCR amplification with primersKS1 and KS4 to yield an approximately 890 bp fragment (“KSFad2-hybrid”,set forth in SEQ ID NO: 72) that was cloned into the commerciallyavailable plasmid pGEM-T Easy (Promega, Madison, Wis.).

The KSFad2 hybrid fragment was then modified to contain additionalrestriction enzyme recognition sites, as follows. The KSFad2 hybridfragment named “KSFad2-hybrid” was re-amplified by standard PCR methodsusing Pfu Turbo DNA polymerase (Stratagene®, La Jolla, Calif.), aplasmid containing KSFad2-hybrid as DNA template, and the followingprimer sets. The oligonucleotide primers (SEQ ID NO: 77 and SEQ ID NO:78) were designed to add a BsiWI restriction endonuclease to the 5′ endof the amplified fragment and to add an AvrII site to its 3′ end. Theresulting DNA “spacer” sequence comprising about 470 nucleotides fromthe soybean Fad2-2 gene and 418 nucleotides from the soybean Fad2-1 isshown in SEQ ID NO: 79.

To prepare the first and second portions of the inverted repeatconstructs, a polynucleotide fragment encoding part of the soybean Lpa1protein (Lpa1, SEQ ID NO: 10) was amplified by standard PCR methodsusing Pfu Turbo® DNA polymerase (Stratagene®, La Jolla, Calif.) and thefollowing primer sets. Lpa1 oligonucleotide primers (SEQ ID NO: 69 andSEQ ID NO: 70) were designed to add NotI and SalI restrictionendonuclease sites at the 5′ end of the amplified fragment and BsiWI andAvrII restriction endonuclease sites at the 3′ end of the amplifiedfragment as well as a stop codon (TAA) at its 3′ end. The DNA sequencecomprising the 556 bp polynucleotide from soybean Lpa1 is set forth inSEQ ID NO: 71.

Preparation of Expression Cassette

An expression cassette was constructed comprising the Lpa1 “IRNTs”operably linked to the strong seed-specific promoter KTI3 (Jofuku et al.(1989) Plant Cell 1: 1079-1093).

A plasmid derived from pKS121 was used to construct the expressioncassette. Plasmid pKS121 was described in PCT Pub. No. WO 02/00904; thisplasmid contains the KTI3 promoter/NotI/Kti3 3′ terminator fragment. Foruse in the present expression cassette, the plasmid pKS121 wasengineered to contain a second hygromycin phosphotransferase gene with a35S-CaMV promoter. The plasmid was then digested with the restrictionenzymes NotI and SalI and the digest was run on a 0.8% TAE-agarose gelto isolate and purify a 7350 bp DNA fragment using the Qiagen® gelextraction kit.

In order to insert the inverted repeat constructs and the spacer regioninto this plasmid, several polynucleotide fragments were prepared.Aliquots of the polynucleotide fragment comprising the 556 bppolynucleotide from soybean Lpa1 (SEQ ID NO: 71) were digested with twoseparate sets of restriction enzymes. First, an aliquot of the amplifiedLpa1 fragment was digested with NotI and BsiWI and run on a 0.8%TAE-agarose gel to isolate a 566 bp DNA fragment, which was purifiedusing the Qiagen® gel extraction kit. A separate aliquot of theamplified Lpa1 fragment was digested with SalI and AvrII and run on a0.8% TAE-agarose gel to isolate a 579 bp DNA fragment, which was alsopurified using the Qiagen® gel extraction kit. Furthermore, theamplified polynucleotide comprising the DNA “spacer” sequence (SEQ IDNO: 79) was digested with BsiWI and AvrII, run on a 0.8% TAE-agarose geland a 901 bp DNA fragment was purified using the Qiagen® gel extractionkit.

To assemble the expression cassette comprising the Lpa1 “IRNTs” operablylinked to the strong seed-specific promoter KTI3, all four isolated andpurified fragments described above were ligated together. The ligationmixture was transformed into E. coli and transformed colonies wereselected on hygromycin. Hygromycin-resistant colonies were selected andgrown overnight in LB media with appropriate antibiotic selection.Proper construction of the expression cassette was confirmed byisolating DNA from these bacterial cultures using a Qiagen® miniprep kitaccording to the manufacturer's protocol and then analyzing withappropriate restriction digests.

Example 12 Production of High P_(i) Lpa1 Transgenic Soybean SomaticEmbryos

The expression cassettes comprising the Lpa1 “IRNTs” operably linked tothe strong seed-specific promoter KTI3 (described in Example 11) wastransformed into soybean embryogenic suspension cultures using theprotocol described in Example 6. Individual immature soybean embryoswere then dried down by transferring them into an empty small Petri dishthat was seated on top of a 10-cm Petri dish containing some agar gel toallow slow dry down. This process is intended to mimic the last stagesof soybean seed development, and dried-down embryos are capable ofproducing plants when transferred to soil or soil-less media. Storageproducts produced by embryos at this stage are similar in composition tostorage products produced by zygotic embryos at a similar stage ofdevelopment and most importantly the storage product profile ispredictive of plants derived from a somatic embryo line (see PCT Pub.No. WO 94/11516).

Determination Inorganic Phosphate Content

Somatic soybean embryos were assayed for P_(i) (inorganic phosphate)content using modifications of Chen et al. ((1956) Anal. Chem. 28:1756-1758). Single embryos were weighed and placed into 1.2 ml deep-welltubes of a 96 well rack (Corning® Incorporated). Metal balls were thenadded to the tubes and the samples were ground using a Geno/Grinder2000™grinder (Sepx CertiPrep®, Metuchen, N.J.). Then 150 μl water was addedto each tube and the rack was shaken for 5 minutes and centrifuged at3,000 g for 5 minutes. The pellet was resuspended and the completeslurry was transferred (without the metal balls) to a new set of into1.2 ml deep-well tubes of a 96 well rack. The original tubes (stillcontaining the metal balls) were washed with an additional 150 μl waterand then shaken for 5 minutes and centrifuged at 2,500 g for 5 minutes.This solution was then pooled with the complete slurry in the new tubes,and 75 μl of 2N HCl was added to each tube. The tubes were incubated for2 hours at room temperature. Thereafter, 188 μl of 30% aqueoustrichloroacetic acid was added to each sample, and the samples weremixed and centrifuged at 2,500 g for 10 minutes. The supernatants weretransferred into fresh tubes and used for P_(i) determinations;measurements were performed in duplicate.

To determine P_(i), 100 μl of each supernatant was placed into a well ofa 96 well microtiter plate and 100 μl of a mixture of 0.42% ammoniummolybdate-1N H₂SO₄:10% ascorbic acid (7:1) was added to each sample. Theplates were incubated at 37° C. for 30 minutes and absorbance wasmeasured at 800 nm; sodium phosphate (NaH₂PO₄) was used as a standard.Table 2 shows data comparing the P_(i) content of transgenic soybeanlines transformed with pJMS33 (described in Example 11) to wild typesomatic embryos. Multiple events were generated expressing the Lpa1 IRNTdescribed in Example 11. Ten out of twenty lines analyzed (50%) showedan increased P_(i) content when compared to wild-type somatic embryos,ranging from 3.5-fold higher than wild type to nearly 8-fold higher thanwild type.

TABLE 2 P_(i) content of somatic soybean embryos from differenttransgenic events expressing the Lpa1 IRNT (as % of wild type (wt)content) Event P_(i) (% of wt) Wild type 100 embryo 4-3 755 3-1 464 4-2350 7-7 432 1-2 496 7-1 520 8-2 759 7-6 381 4-1 543 8-3 478

Example 13 Transgenic Maize Seeds have Reduced Phytic Acid and IncreasedP_(i) Content

Two expression cassettes were constructed to provide cosuppression of anMRP. These expression cassettes (designated plasmids P36 and P94) weremade using MRP polynucleotide fragments. Each construct contained aninverted repeat of an MRP polynucleotide such that the first and secondportions self-hybridized to produce a hairpin structure that cansuppress expression of the relevant endogenous gene (e.g., maize Lpa1).Between the two fragments of the inverted repeat was an intron thathelped to form the loop portion in the hairpin structure. Transcriptionwas driven by the oleosin promoter in plasmid P36 and by the Glb1promoter in plasmid P94; neither construct had a terminator. In bothplasmids P36 and P94, the intron used was the Adh1 intron (GenBankAccession No. X04050), although other introns may also be used.

The plasmids were used to produce transgenic maize using protocolsdescribed in Example 1. Transgenic T1 seeds were screened for elevatedP_(i) content using a rapid P_(i) assay, and quantitative analysis ofphytic acid and P_(i) were also performed. The results of these assaysdemonstrated that cosuppression of MRP expression resulted in a decreasein phytic acid content and an increase in P_(i) in the transgenic seeds(see Table 3).

TABLE 3 Maize plants transformed with MRP expression constructs producedtransgenic seeds with reduced phytic acid and increased P_(i) content.Wt K CS K Plasmid #/ Wt K P_(i) CS K P_(i) PAP PAP PA Event (mg/g)(mg/g) (mg/g) (mg/g) reduction Plasmid 36 1 0.31 1.17 2.76 0.72 74% 20.18 1.05 2.75 0.73 74% 3 0.27 0.99 2.53 0.99 61% 4 0.26 1.21 2.43 0.8466% 5 0.43 1.12 2.15 0.85 60% 6 0.31 1.20 2.41 0.79 67% 7 0.34 1.06 2.590.61 77% 8 0.26 1.15 2.60 0.57 78% 9 0.21 1.09 2.61 0.70 73% 10  0.311.26 2.55 0.82 68% 11  0.19 1.08 2.46 0.66 73% 12  0.32 1.09 2.50 0.7869% Plasmid 94 1 0.14 1.01 3.47 2.29 34% 2 0.12 1.37 3.10 1.12 64% 30.16 1.44 3.09 1.00 68% 4 0.10 1.20 3.44 1.75 49% 5 0.24 1.25 3.04 1.5350% 6 0.24 1.47 2.67 0.98 63% 7 0.21 1.46 2.98 1.11 63% 8 0.18 1.17 3.001.76 41% Wt K = wild-type kernels in a segregation ear; CS K =cosuppression kernels in a segregation ear; P_(i) = inorganic phosphatephosphorus; PAP = phytic acid phosphorus; PA = phytic acid

As indicated in the table legend, “Wt K” were kernels in a segregationear without the MRP transgene and “CS K” were the kernels in the samesegregation ear that did contain the MRP transgene. The PAP values inTable 3 were measured according to modifications of the methodsdescribed by Latta & Eskin (1980) J. Agric Food Chem. 28: 1313-1315 andVaintraub & Lapteva (1988) Analytical Biochemistry 175: 227-230; seeExample 1 for detail.

Example 14 Production of Transgenic Sorghum

The promoter construct prepared in Example 10 is used to transformsorghum according to the teachings of U.S. Pat. No. 6,369,298. Briefly,a culture of Agrobacterium is transformed with a vector comprising anexpression cassette containing the promoter construct prepared inExample 10. The vector also comprises a T-DNA region into which thepromoter construct is inserted. General molecular techniques used in theinvention are provided, for example, by Sambrook et al. (eds.) MolecularCloning: A Laboratory Manual, 1989, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.

Immature sorghum embryos are obtained from the fertilized reproductiveorgans of a mature sorghum plant. Immature embryos are asepticallyisolated from the developing kernel at about 5 days to about 12 daysafter pollination and held in sterile medium until use; generally, theembryos are about 0.8 to about 1.5 mm in size.

The Agrobacterium-mediated transformation process of the invention canbe broken into several steps. The basic steps include: an infection step(step 1); a co-cultivation step (step 2); an optional resting step (step3); a selection step (step 4); and a regeneration step (step 5). In theinfection step, the embryos are isolated and the cells contacted withthe suspension of Agrobacterium.

The concentration of Agrobacterium used in the infection step andco-cultivation step can affect the transformation frequency. Very highconcentrations of Agrobacterium may damage the tissue to be transformed,such as the immature embryos, and result in a reduced callus response.The concentration of Agrobacterium used will vary depending on theAgrobacterium strain utilized, the tissue being transformed, the sorghumgenotype being transformed, and the like. Generally a concentrationrange of about 0.5×10⁹ cfu/ml to 1×10⁹ cfu/ml will be used.

The embryos are incubated with the suspension of Agrobacterium about 5minutes to about 8 minutes. This incubation or infection step takesplace in a liquid solution that includes the major inorganic salts andvitamins of N6 medium (referred to as “N6 salts,” or medium containingabout 463.0 mg/l ammonium sulfate; about 1.6 mg/l boric acid; about 125mg/l calcium chloride anhydrous; about 37.25 mg/l Na₂-EDTA; about 27.8mg/l ferrous sulfate.7H₂O; about 90.37 mg/l magnesium sulfate; about3.33 mg/l manganese sulfate H₂O; about 0.8 mg/l potassium iodide; about2,830 mg/l potassium nitrate; about 400 mg/l potassium phosphatemonobasic; and about 1.5 mg/l zinc sulfate.7 H₂O.

In addition, the media in the infection step generally excludes AgNO₃.AgNO₃ is generally included in the co-cultivation, resting (when used)and selection steps when N6 media is used. In the co-cultivation step,the immature embryos are co-cultivated with the Agrobacterium on a solidmedium. The embryos are positioned axis-down on the solid medium and themedium can include AgNO₃ at a range of about 0.85 to 8.5 mg/l. Theembryos are co-cultivated with the Agrobacterium for about 3-10 days.

Following the co-cultivation step, the transformed cells may besubjected to an optional resting step. Where no resting step is used, anextended co-cultivation step may utilized to provide a period of culturetime prior to the addition of a selective agent. For the resting step,the transformed cells are transferred to a second medium containing anantibiotic capable of inhibiting the growth of Agrobacterium. Thisresting phase is performed in the absence of any selective pressures onthe plant cells to permit preferential initiation and growth of callusfrom the transformed cells containing the heterologous nucleic acid. Theantibiotic added to inhibit Agrobacterium growth may be any suitableantibiotic; such antibiotics are known in the art and includeCefotaxime, timetin, vancomycin, carbenicillin, and the like.Concentrations of the antibiotic will vary according to what is standardfor each antibiotic, and those of ordinary skill in the art willrecognize this and be able to optimize the antibiotic concentration fora particular transformation protocol without undue experimentation. Theresting phase cultures are preferably allowed to rest in the dark at 28°C. for about 5 to about 8 days. Any of the media known in the art can beutilized for the resting step.

Following the co-cultivation step, or following the resting step, whereit is used, the transformed plant cells are exposed to selectivepressure to select for those cells that have received and are expressingpolypeptide from the heterologous nucleic acid introduced byAgrobacterium. Where the cells are embryos, the embryos are transferredto plates with solid medium that includes both an antibiotic to inhibitgrowth of the Agrobacterium and a selection agent. The agent used toselect for transformants will select for preferential growth of explantscontaining at least one selectable marker insert positioned within thesuperbinary vector and delivered by the Agrobacterium. Generally, any ofthe media known in the art suitable for the culture of sorghum can beused in the selection step, such as media containing N6 salts or MSsalts. During selection, the embryos are cultured until callus formationis observed. Typically, calli grown on selection medium are allowed togrow to a size of about 1.5 to about 2 cm in diameter.

After the calli have reached the appropriate size, the calli arecultured on regeneration medium in the dark for several weeks to allowthe somatic embryos to mature, generally about 1 to 3 weeks. Preferredregeneration media includes media containing MS salts. The calli arethen cultured on rooting medium in a light/dark cycle until shoots androots develop. Methods for plant regeneration are known in the art (see,e.g., Kamo et al. (1985) Bot. Gaz. 146(3): 327-334; West et al. (1993)Plant Cell 5:1361-1369; and Duncan et al. (1985) Planta 165: 322-332).

Small plantlets are then transferred to tubes containing rooting mediumand allowed to grow and develop more roots for approximately anotherweek. The plants are then transplanted to soil mixture in pots in thegreenhouse.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claim(s).

That which is claimed:
 1. An isolated nucleic acid molecule comprising anucleotide sequence that encodes a polypeptide that modulates the levelof phytate in a plant, wherein the nucleotide sequence is selected fromthe group consisting of: a) a nucleotide sequence which has at least 90%sequence identity to the sequence set forth in nucleotides 244-4776 ofSEQ ID NO: 2; b) a nucleotide sequence which encodes a polypeptidehaving an amino acid sequence that shares at least 93% sequence identitywith the amino acid sequence set forth in SEQ ID NO: 3 or 5; c) anucleotide sequence which has at least 90% sequence identity to thesequence set forth in nucleotides 245-4762 of SEQ ID NO: 6; d) anucleotide sequence which encodes a polypeptide having an amino acidsequence that shares at least 90% sequence identity with the amino acidsequence set forth in SEQ ID NO: 7; e) a nucleotide sequence which hasat least 90% sequence identity to the sequence set forth in nucleotides3-1350 of SEQ ID NO: 10; f) a nucleotide sequence which encodes apolypeptide having an amino acid sequence that shares at least 90%sequence identity with the amino acid sequence set forth in SEQ ID NO:11; g) a nucleotide sequence which has at least 90% sequence identity tothe sequence set forth in nucleotides 1-465 of SEQ ID NO: 12; h) anucleotide sequence which encodes a polypeptide having an amino acidsequence that shares at least 90% sequence identity with the amino acidsequence set forth in SEQ ID NO: 13; i) a nucleotide sequence which hasat least 90% sequence identity to the sequence set forth in SEQ ID NO:71; and j) a nucleotide sequence which encodes a polypeptide having anamino acid sequence that comprises the sequence set forth in SEQ ID NO:15.
 2. The nucleic acid molecule of claim 1, wherein said nucleotidesequence encodes a polypeptide comprising an amino acid sequence havingat least 95% sequence identity to the amino acid sequence set forth inSEQ ID NO:
 3. 3. The nucleic acid molecule of claim 2, wherein saidnucleotide sequence encodes a polypeptide comprising the amino acidsequence set forth in SEQ ID NO:
 3. 4. An expression cassette comprisingthe nucleic acid molecule of claim 1, wherein said nucleotide sequenceis operably linked to a promoter that drives expression in amicroorganism or in a plant cell.
 5. An isolated polypeptide comprisingan amino acid sequence which has at least 93% sequence identity to theamino acid sequence set forth in SEQ ID NO: 3, wherein said polypeptidemodulates the level of phytate in a plant.
 6. An expression cassettecomprising a first nucleotide sequence selected from the groupconsisting of: a) a nucleotide sequence having at least 90% sequenceidentity to a nucleotide sequence comprising at least 50 contiguousnucleotides of the nucleotide sequence set forth in SEQ ID NO: 1, 2, 4,6, 8, 10, 12, 71, or 14; b) a nucleotide sequence comprising at least 19contiguous nucleotides of the nucleotide sequence set forth in SEQ IDNO: 1, 2, 4, 6, 8, 10, 12, 71, or 14; c) a nucleotide sequence encodingan amino acid sequence that has at least 90% sequence identity to theamino acid sequence set forth in SEQ ID NO: 3, 5, 7, 9, 11, 13, or 15;and d) a nucleotide sequence which is the complement of a), b), or c).7. A method for producing food or feed with a reduced level of phytate,said method comprising: a) transforming a plant with a nucleic acidmolecule comprising a first nucleotide sequence selected from the groupconsisting of: i) a nucleotide sequence having at least 90% sequenceidentity to a nucleotide sequence comprising at least 50 contiguousnucleotides of the nucleotide sequence set forth in SEQ ID NO: 1, 2, 4,6, 8, 10, 12, 71, or 14; ii) a nucleotide sequence comprising at least19 contiguous nucleotides of the nucleotide sequence set forth in SEQ IDNO: 1, 2, 4, 6, 8, 10, 12, 71, or 14; iii) a nucleotide sequenceencoding an amino acid sequence that has at least 90% sequence identityto the amino acid sequence set forth in SEQ ID NO: 3, 5, 7, 9, 11, 13,or 15; and iv) a nucleotide sequence which is the complement of i), ii),or iii); b) growing said plant under conditions in which said nucleotidesequence is expressed; and c) producing food or feed from said plant,wherein said plant has a reduced level of phytate in comparison to acontrol plant.
 8. The method of claim 7, wherein said first nucleotidesequence has at least 95% sequence identity to the nucleotide sequenceset forth in nucleotides 244-4776 of SEQ ID NO:
 2. 9. The method ofclaim 7, wherein said plant is further transformed with a nucleic acidmolecule comprising a second nucleotide sequence selected from the groupconsisting of: a) a nucleotide sequence having at least 90% sequenceidentity to a nucleotide sequence comprising at least 50 contiguousnucleotides of the nucleotide sequence set forth in SEQ ID NO: 1, 2, 4,6, 8, 10, 12, 71, or 14; b) a nucleotide sequence comprising at least 19contiguous nucleotides of the nucleotide sequence set forth in SEQ IDNO: 1, 2, 4, 6, 8, 10, 12, 71, or 14; c) a nucleotide sequence encodingan amino acid sequence that has at least 90% sequence identity to theamino acid sequence set forth in SEQ ID NO: 3, 5, 7, 9, 11, 13, or 15;and d) a nucleotide sequence which is the complement of i), ii), oriii); wherein said plant has a reduced level of phytate in comparison toa control plant.
 10. The method of claim 7, wherein said plant isfurther transformed with a nucleic acid molecule comprising a secondnucleotide sequence selected from the group consisting of: a) an mi1psnucleotide sequence; b) an IPPK nucleotide sequence; c) an ITPK-5nucleotide sequence; d) an IP2K nucleotide sequence; e) an MIKnucleotide sequence; f) a phytase nucleotide sequence; g) a nucleotidesequence having at least 90% sequence identity to the nucleotidesequence set forth in SEQ ID NO: 25, 64, 65, 67, or 68; h) a nucleotidesequence comprising at least 19 nucleotides of the sequence set forth inSEQ ID NO: 25, 64, 65, 67, or 68; i) a nucleotide sequence which is thecomplement of (a), (b), (c), (d), (e), (g), or (h); and j) a nucleotidesequence having at least 90% sequence identity to the nucleotidesequence set forth in SEQ ID NO:
 66. 11. The method of claim 7, whereinsaid plant is further transformed with a nucleic acid moleculecomprising a second nucleotide sequence conferring a trait of intereston said transformed plant.
 12. The method of claim 11, wherein saidtrait of interest is selected from the group consisting of: a) high oil;b) increased digestibility; c) high energy; d) balanced amino acid; e)high oleic acid; f) insect resistance; g) disease resistance; h)herbicide resistance; i) drought tolerance; and j) male sterility.
 13. Atransformed plant comprising in its genome at least one stablyincorporated nucleic acid molecule having a first nucleotide sequenceselected from the group consisting of: a) a nucleotide sequence havingat least 90% sequence identity to a nucleotide sequence comprising atleast 50 contiguous nucleotides of the nucleotide sequence set forth inSEQ ID NO: 1, 2, 4, 6, 8, 10, 12, 71, or 14; b) a nucleotide sequencehaving at least 90% sequence identity to the nucleotide sequence setforth in SEQ ID NO: 1, 2, 4, 6, 8, 10, 12, 71, or 14; c) a nucleotidesequence comprising at least 19 nucleotides of the sequence set forth inSEQ ID NO: 1, 2, 4, 6, 8, 10, 12, 71, or 14; and d) a nucleotidesequence which is the complement of a), b), or c); wherein said planthas a reduced level of phytate compared to a control plant.
 14. Thetransformed plant of claim 13, wherein said plant is further transformedwith a nucleic acid molecule comprising a second nucleotide sequenceselected from the group consisting of: a) an mi1ps nucleotide sequence;b) an IPPK nucleotide sequence; c) an ITPK-5 nucleotide sequence; d) anIP2K nucleotide sequence; e) an MIK nucleotide sequence; f) a phytasenucleotide sequence; g) a nucleotide sequence having at least 90%sequence identity to the nucleotide sequence set forth in SEQ ID NO: 25,64, 65, 67, or 68; h) a nucleotide sequence comprising at least 19nucleotides of the sequence set forth in SEQ ID NO: 25, 64, 65, 67, or68; i) a nucleotide sequence which is the complement of (a), (b), (c),(d), (e), (g) or (h); and j) a nucleotide sequence having at least 90%sequence identity to the nucleotide sequence set forth in SEQ ID NO: 66.15. The transformed plant of claim 13, wherein said plant is furthertransformed with a nucleic acid molecule comprising at least one secondnucleotide sequence that confers at least one trait of interest on saidtransformed plant.
 16. The transformed plant of claim 15, wherein saidtrait of interest is selected from the group consisting of: a) high oil;b) increased digestibility; c) high energy; d) balanced amino acidcomposition; e) high oleic acid; f) insect resistance; g) diseaseresistance; h) herbicide resistance; i) drought tolerance; and j) malesterility.
 17. Transformed seed of the plant of claim 13, wherein saidseed comprises said first nucleotide sequence.
 18. Food or feedcomprising the plant of claim
 13. 19. Food or feed comprising thetransformed seed of claim
 13. 20. The transformed plant of claim 13,wherein said plant is a monocot.
 21. A method for producing food or feedwith a reduced level of phytate, said method comprising the steps of:(a) transforming a plant cell with at least one first polynucleotidecomprising at least 19 nucleotides of the sequence set forth in SEQ IDNO: 1, 2, 4, 6, 8, 10, 12, or 71; (b) transforming a plant cell with atleast one second polynucleotide having at least 94% sequence identity tothe complement of the polynucleotide of step (a); (c) regenerating atransformed plant from said plant cell; and (d) producing food or feedfrom said transformed plant or from seed of said transformed plant;wherein the level of phytate in said plant is reduced in comparison to acontrol plant.
 22. A plant containing an Lpa1 insertion mutationcomprising a Mu element, wherein said Mu element is inserted in the Lpa1gene in a location selected from the group consisting of: a) in exon 1;b) at nucleotide 585 in exon 1; c) at nucleotide 874 in exon 1; d) inexon 11; and e) at nucleotide 6069 in exon 11.